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THE  LIBRARY 

OF 

THE  UNIVERSITY 

OF  CALIFORNIA 

LOS  ANGELES 


GIFT 
Dr.  Oustav  F.  Ruediger 


CONTRIBUTIONS  TO  MEDICAL  SCIENCE 


THE  UNIVERSITY  OF  CHICAGO  PRESS 
CHICAGO.  ILLINOIS 

Bgents 
THE  BAKER  &  TAYLOR  COMPANY 

NEW    TOBK 

CAMBRIDGE  UNIVERSITY  PRESS 

LONDON    AND    EDINBURGH 


'/^  .~r<jlc<i^fsk~^ 


CONTRIBUTIONS 

TO  MEDICAL  SCIENCE  BY 

HOWARD  TAYLOR 

RICKETTS 

187O-I9IO 


PUBLISHED 

AS  A  TRIBUTE  TO  HIS  MEMORY  BY  HIS  COLLEAGUES 

UNDER  THE  AUSPICES  OF  THE  CHICAGO 

PATHOLOGICAL  SOCIETY 


THE  UNIVERSITY  OF  CHICAGO  PRESS 
CHICAGO,   ILLINOIS 


Copyright  igii  By 
The  University  of  Chicago 


All  Rights  Reserved 


Published  May  igii 


Composed  and  Printed  By 

The  University  of  Chicago  Press 

Chicago,  Illinois,  U.S.A. 


INTRODUCTORY  NOTE 

Shortly  after  the  death  of  Dr.  Ricketts  the  Chicago  Pathological 
Society  appointed  a  committee  to  prepare  a  suitable  memorial. 

On  account  of  the  great  importance  of  Dr.  Ricketts'  work  in 
blastomycosis,  in  Rocky  Mountain  spotted  fever,  and  in  the 
typhus  fever  of  Mexico  City,  it  seemed  that  the  pubHcation  of  his 
articles  in  permanent  form  would  be  a  useful  tribute. 

The  chief  significance  of  this  volume  as  a  memorial  lies  in 
the  circumstance  that  the  cost  of  its  pubHcation  is  borne  by  Dr. 
Ricketts'  friends  and  admirers  in  the  various  parts  and  laborato- 
ries of  the  country. 

The  repubhshed  articles  are  printed  in  their  original  form, 
the  only  changes  being  those  incidental  to  new  pagination,  etc. 

Several  articles  by  collaborators  and  pupils  of  Dr.  Ricketts 
have  been  included  because  they  were  inspired  by  him  and  were 
in  course  of  preparation  before  his  death. 

It  did  not  seem  advisable  to  include  the  following  papers  by 
Dr.  Ricketts:  "A  Case  of  Diplococcemia  Characterized  by 
Repeated  Attacks  of  Fever,  Muscular  Pains,  and  an  Erythematous 
Eruption"  (with  Lewellys  F.  Barker),  Trans.  Chic.  Path.  Soc, 
1903,  5,  p.  313;  "Our  Serotherapeutic  Measures,"  Jour.  Am. 
Med.  Assn.,  1904,  42,  p.  1336;  "Fundamental  Principles  of  Immu- 
nity," Am.  Jour.  Obs.,  1905,  52,  p.  801;  "Recent  Studies  of  Rocky 
Mountain  Fever  in  Montana  and  Idaho,"  Med.  Sentinel,  1908, 
16,  p.  666;  "General  Report  of  an  Investigation  of  Rocky  Moun- 
tain Spotted  Fever,  Carried  on  during  1906  and  1907,"  a  report  to 
the  Montana  State  Board  of  Health;  and  the  weekly  articles 
on  immunity  in  the  Journal  of  the  American  Medical  Association 
out  of  which  grew  the  book  on  Infection,  Immunity,  and  Serum 
>^  Therapy    (Chicago,    1906).     Two  pathological  reports  on  cases 

-!-^  V 


WJt'OSO 


vi  Introductory  Note 

of  blastomycosis  and  certain  notes  of  a  strictly  preliminary  nature 

have  been  omitted  also,  but  references  to  them  are  inserted  in 

the  proper  places. 

LuDviG  Hektoen 

Preston  Kyes 

E.  R.  Le  Count 

George  H.  Weaver 

H.  Gideon  Wells 

Committee 


TABLE   OF   CONTENTS 

PAGE 

Introductory  Note v 

Howard  Taylor  Ricketts 3 

LuDviG  Hektoen. 


A  New  Mould-Fungus  as  the  Cause  of  Three  Cases  of  So- 

Called  Blastomycosis  or  Oidiomycosis  of  the  Skin      .       ii 
H.  T.  Ricketts. 

Oidiomycosis     (Blastomycosis)   of  the  Skin  and  Its  Fungi      i8 
H.  T.  Ricketts. 

An  Organism  from  Cutaneous  Oidiomycosis  (Blastomycosis). 

Remarks  on  Classification 176 

H.  T.  Ricketts. 

The  Immunological  Reactions  of  Oidiomycosis  (Blastomy- 
cosis)  IN  THE   GuiNEA-PiG 1 79 

Benj.  F.  Davis. 

Lymphatotoxic  Serum:  Notes  on  Its  Constitution;  Pre- 
liminary  Experiments   Bearing   on   Its   Influence   on 

Experimental  Infections 231 

H.  T.  Ricketts. 

Preliminary  Report  on  the  Action  of  Neurotoxic  Serum    240 
H.  T.  Ricketts  and  T.  Rothstein. 

The  Reduction  of  Methylene  Blue  by  Nervous  Tissue  245 

H.  T.  Ricketts. 

Concerning  the  Possibility  of  an  Antibody  for  the  Tetan- 

ophile  Receptor  of  Erythrocytes:    A  Receptor  Study    254 
H.  T.  Ricketts. 

The  Adjuvant  Action  of  Serum,  Egg-Albumin,  and  Broth  on 

Tetanus  Intoxication 266 

H.  T.  Ricketts  A>rD  E.  J.  Kirk. 

The  Study  of  "Rocky  Mountain  Spotted  Fever"  (Tick 
Fever  ?)  by  Means  of  Animal  Inoculations.  A  Prelimi- 
nary Communication 278 

H.  T.  Ricketts. 


viii  Table  of  Contents 

PAGE 

The  Transmission  of  Rocky  Mountain  Spotted  Fever  by 

THE  Bite  of  the  Wood-Tick  (Dermacentor  occidentalis)     288 

H.    T.    RiCKETTS. 

Further  Observations  on  Rocky  Mountain  Spotted  Fever 

and  Dermacentor  Occidentalis 291 

H.    T.    RiCKETTS. 

Observations  on  the  Virus  and  Means  of  Transmission  of 

Rocky  Mountain  Spotted  Fever 299 

H.    T.    RiCKETTS. 

The  R6le  of  the  Wood-Tick  (Dermacentor  occidentalis) 
IN  Rocky  Mountain  Spotted  Fever,  and  the  Suscepti- 
bility OF  Local  Animals  to  This  Disease.  A  Prelimi- 
nary Report 312 

H.    T.    RiCKETTS. 

Further  Experiments  with  the  Wood-Tick  in  Relation  to 

Rocky  Mountain  Spotted  Fever 324 

H.    T.    RiCKETTS. 

A  Summary  of  Investigations  of  the  Nature  and  Means  of 

Transmission  of  Rocky  Mountain  Spotted  Fever  .       .     333 

H.   T.    RiCKETTS. 

Studies  on  Immunity  in  Rocky  Mountain  Spotted  Fever  343 

H.  T.  RiCKETTS  and  L.  Gomez. 

A  Micro-organism  Which  Apparently  Has  a  Specific  Rela- 
tionship to  Rocky  Mountain  Spotted  Fever.  A  Prelimi- 
nary Report 368 

H.    T.    RiCKETTS. 

Some  Aspects  of  Rocky  Mountain  Spotted  Fever  as  Shown 
BY  Recent  Investigations  (The  Wesley  M.  Carpenter 
Lecture  of  the  New  York  Academy  of  Medicine,  1909)   .  373 

H.   T.    RiCKETTS. 

Unfinished  Experiments  of  Dr.  Howard  T.  Ricketts  on 

Rocky  Mountain  Spotted  Fever 409 

Benj.  F.  Davis. 

Complement  Deviation  in  Rocky  Mountain  Spotted  Fever         419 
Benj,  F.  Davis  and  William  F.  Petersen. 


Table  of  Contents  ix 

PAGE 

Time  Relationships  of  the  Wood-Tick  in  the  Transmission 

OF  Rocky  Mountain  Spotted  Fever 428 

JosiAH  J.  Moore. 

Transmission  of  Spotted  Fever  by  the  Tick  in  Nature  437 

Maria  B.  Maver. 

Transmission  of  Spotted  Fever  by  Other  than  Montana  and 

Idaho  Ticks 44° 

Maria  B.  Maver. 

A  Contribution  to  the  Pathological  Anatomy  of  Rocky 

Mountain  Spotted  Fever 445 

E.  R.  Le  Count. 

The  Typhus  Fever  of  Mexico  (Tabardillo).    Preliminary 

Observations 451 

H.  T.  Ricketts  and  Russell  M.  Wilder. 

The  Transmission  of  the  Typhus  Fever  of  Mexico  (Tabar- 
dillo) BY  Means  of  the  Louse  (Pediculus  vestimenti)   .     463 
H.  T.  Ricketts  and  Russell  M.  Wilder. 

The  Etiology  of  the  Typhus  Fever  (Tabardillo)  of  Mexico 

City.    A  Further  Preliminary  Report       ....     473 
H.  T.  Ricketts  and  Russell  M.  Wilder. 

The  Relation  of  Typhus  Fever  (Tabardillo)  to  Rocky  Moun- 
tain Spotted  Fever 479 

H.  T.  Ricketts  and  Russell  M.  Wilder. 

Further  Investigations  Regarding  the  Etiology  of  Tabar- 
dillo, Mexican  Typhus  Fever 491 

H.  T.  Ricketts  and  Russell  M.  Wilder. 


HOWARD  TAYLOR  RICKETTS 


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HOWARD  TAYLOR  RICKETTS.^ 

LuDviG   Hektoen. 

Dr.  Ricketts  was  born  in  Findlay,  Ohio,  February  g,  187 1, 
passed  his  boyhood  in  Nebraska,  and  received  the  Bachelor's 
degree  from  the  university  of  that  state  in  1894.  He  took  his 
medical  course  at  the  Northwestern  University  Medical  School, 
graduating  in  1897,  and  then  served  as  interne  in  the  Cook  County 
Hospital  in  Chicago.  In  1899  he  became  fellow  in  cutaneous 
pathology  in  Rush  Medical  College  and  continued  as  such  for  two 
years.  During  part  of  this  time  he  worked  also  in  the  dermato- 
logical  clinic.  In  1900  he  married  Myra  Tubbs  who  supported 
him  in  his  work  with  rare  devotion,  keen  interest,  and  steady 
encouragement.     There  are  two  children,  son  and  daughter. 

In  1902,  returning  from  a  year's  visit  to  European  laboratories, 
he  came  to  the  University  of  Chicago  as  instructor  in  the  newly 
founded  Department  of  Pathology  and  Bacteriology.  Later  he 
became  assistant  professor  and  in  the  early  part  of  19 10  he  accepted 
the  chair  of  pathology  in  the  University  of  Pennsylvania,  the 
duties  of  which  he  fully  expected  to  assume  in  the  fall.  He  died 
from  typhus  fever  in  Mexico  City,  May  3,  1910. 

Dr.  Ricketts  was  a  modest  and  unassuming  man,  of  great 
determination  and  of  the  highest  character,  loyal  and  generous, 
earnest  and  genuine  in  all  his  doings — a  personahty  of  unusual 
and  winning  charm.  His  associates  of  the  hospital  and  fellowship 
days,  who  knew  him  well,  knew  his  abihty  and  energy,  his  distinct 
fondness  for  the  day's  work,  all  looked  to  him  for  the  more  than 
ordinary  achievement. 

He  deUberately  turned  away  from  the  allurements  of  active 
medical  practice  and  decided  to  devote  himself  to  teaching  and 
investigation  in  pathology.  He  had  early  become  possessed  of 
noble  ideals  and  had  a  pure  love  for  the  search  after  truth  in  his 
chosen  field,  which  abided  with  him  and  gave  him  a  high  concep- 
tion of  all  his  duties  and  relations  and  placed  a  special  stamp  on 

>  An  address  delivered  at  a  memorial  service  in  the  University  of  Chicago,  May  15,  1910,  and  now 
expanded  to  include  a  few  more  details. 

3 


4  Contributions  to  Medical  Science 

his  work.  His  instinct  for  research  at  no  time  was  permitted  to 
lie  dormant  and  unused,  but  growing  stronger  it  carried  him  on 
farther  and  farther,  and  in  due  time  the  University  freely  and  in 
special  ways  promoted  the  work  in  which  he  was  to  accomplish 
such  large  results.  The  torch  was  placed  within  the  grasp  of  hands 
fit  to  carry  it  forward,  and  during  the  few  short  years  given  him 
he  advanced  it  farther  than  we  may  realize  at  this  moment,  because 
he  broke  open  paths  for  future  progress. 

His  earher  researches  on  blastomycosis  and  immunological 
problems  are  all  marked  by  thoroughness  and  directness,  by  clear 
and  forceful  reasoning;  it  is  in  the  brilliant  work  on  Rocky  Moun- 
tain fever,  however,  that  Dr.  Ricketts  fully  reveals  himself  as  an 
investigator  of  the  first  rank.  He  took  up  the  study  of  this  fever 
in  the  spring  of  1906  as  a  sort  of  pastime  during  an  enforced  hoHday 
on  account  of  overwork.  The  disease  is  a  remarkable  one;  it 
occurs  in  well-defined  areas  in  the  Mountains,  is  sharply  limited 
to  the  spring  months,  varies  greatly  in  severity,  the  mortality  in 
one  place  being  about  5,  in  another  between  80  and  90,  per  cent. 
For  some  time  it  had  been  regarded  as  caused  in  some  way  by  the 
bite  of  a  tick.  Dr.  Ricketts  promptly  found  that  the  disease  is 
communicable  to  lower  animals  and  that  a  certain  tick,  which 
occurs  naturally  on  a  large  number  of  animals  in  those  regions, 
by  its  bite  can  transmit  the  disease  from  the  sick  to  the  healthy 
animal. 

These  observations  opened  a  new  field,  and  henceforth  he 
devoted  himself  untiringly  to  the  investigation  of  the  many  prob- 
lems that  arose  one  after  another  as  the  work  went  on,  both  in  the 
laboratory  here  and  in  the  field.  As  we  follow  the  various  stages 
in  the  progress  of  this  intensely  active  work  it  becomes  very  clear 
that  Dr.  Ricketts  not  only  was  gifted  with  imaginative  power  so 
that  he  could  see  and  trace  the  various  fines  along  which  the 
solution  of  a  problem  might  be  sought,  but  that  he  also  possessed 
in  a  full  measure  the  capacity  for  that  hard,  accurate,  patient  work 
necessary  for  the  more  dijficult  task  of  finding  the  one,  true  solution. 
This  combination  of  speculative  abihty  and  the  power  to  do 
steady  toil  and  even  drudgery,  often  under  great  difficulties,  made 
him  a  great  investigator  and  brought  him  success. 


Howard  Taylor  Ricketts  S 

Some  of  the  experiments  devised  to  lay  bare  the  secrets  of  the 
different  orders  of  living  things  concerned  in  spotted  fever  are 
masterful  in  their  ingenuity  and  comprehensiveness,  notably  those 
bearing  on  the  hereditary  transmission  of  spotted  fever  virus  in 
ticks,  on  the  occurrence  of  mfected  ticks  in  nature,  and  on  the  part 
played  by  small  wild  animals  like  the  squirrel  as  source  for  the 
virus. 

Having  solved  many  hard  questions  he  came  to  the  conclusion 
that  in  man  spotted  fever  depends  simply  on  the  accidental  bite 
by  an  adult  tick  carrying  active  virus.  As  only  adult  ticks  find 
their  way  to  man  and  as  they  occur  only  in  the  spring,  the  pecuHar 
seasonal  prevalence  of  the  disease  is  nicely  explained.  It  is  almost 
unnecessary  to  point  out  that  the  work  furnishes  clear  and  direct 
indications  as  to  what  to  do  in  order  to  prevent  the  disease.  Finally 
last  year,  he  discovered  what  seems  to  be  the  immediate  cause  of 
spotted  fever,  namely  a  small  bacillus,  which  he  found  in  the  blood 
of  patients  and  in  ticks  and  their  eggs.  Strains  of  this  bacillus 
present  in  ticks  from  different  places  vary  greatly  in  virulence,  and 
this  fact  may  explain  why  spotted  fever  varies  so  greatly  in  severity. 

Many  of  the  observations  and  discoveries  in  connection  with 
this  work  have  a  much  wider  significance,  and  will  surely  prove  of 
value  and  service  on  the  ever-shifting  battleground  with  infectious 
diseases. 

Rocky  Mountain  spotted  fever  in  many  ways  resembles  typhus 
fever.  As  he  was  completing  his  three  years'  study  of  the  Rocky 
Mountain  disease,  having  determined  its  mode  of  transmission,  its 
cause,  and  a  rational  method  for  its  prevention.  Dr.  Ricketts 
became  more  and  more  strongly  impressed  with  the  thought,  which 
he  had  had  for  some  time,  that  the  special  knowledge  and  training 
thus  acquired  would  prove  of  great  value  in  the  study  of  typhus 
fever  and  thereby  perhaps  be  put  to  the  best  use.  This  idea  met 
with  encouragement,  and  in  July  of  last  year  it  was  definitely 
decided  to  take  up  the  study  of  typhus  fever  in  the  City  of  Mexico, 
that  being  the  nearest  place,  so  far  as  known,  where  any  such 
work  could  be  done.  I  speak  of  this  date  because  I  wish  to  make 
it  clear  that  Dr.  Ricketts  reached  his  decision  before,  and  entirely 
independently  of,  the  establishment  by  the  Mexican  government 


6  Contributions  to  Medical  Science 

of  certain  prizes  for  successful  investigation  of  the  typhus  fever 
of  Mexico  (Tabardillo). 

Typhus  fever  has  been  one  of  the  great  epidemic  diseases  of  the 
world.  Its  devastations  are  recorded  ''on  the  dark  pages  of 
history,  the  pages  that  tell  of  war,  overcrowding,  want,  and  misery." 
Until  the  middle  of  the  last  century  it  prevailed  in  practically  all 
large  European  cities;  now  it  has  largely  disappeared,  owing,  it 
is  believed,  to  better  sanitary  conditions;  but  it  is  still  smoldering 
in  many  centers,  and  in  some  places,  as  in  Mexico,  typhus  in  one 
of  its  forms  now  claims  hundreds  of  victims  each  year.  When  it 
assumes  its  most  virulent  forms  typhus  fever  may  become  one  of 
the  most  contagious  of  diseases,  and  there  is  no  disease  that  has 
claimed  so  many  victims  among  physicians  and  nurses.  It  is 
stated  that  in  a  period  of  twenty-five  years,  of  1,230  physicians 
attached  to  institutions  in  Ireland  550  succumbed  to  typhus.  Of 
the  six  American  scientists  who  have  studied  the  typhus  fever 
of  Mexico  since  December  last,  three  have  been  stricken  and  two 
have  died — Conneff  of  the  Ohio  State  University  expedition  and 
our  Ricketts.  It  is  when  the  sick  are  aggregated  in  hospital  wards 
that  the  danger  of  infection  is  especially  great.  Until  very  recently 
nothing  was  known  as  to  the  cause  of  typhus  fever  and  the  exact 
mode  of  its  transmission. 

Fully  acquainted  as  a  matter  of  course  with  all  the  character- 
istics of  the  disease,  Dr.  Ricketts  and  his  volunteer  assistant,  Mr. 
Russell  M.  Wilder,  began  their  work  in  December  last.  Before 
many  weeks  had  passed  results  of  great  importance  were  secured; 
it  was  found  that  tophus  is  different  from  the  Rocky  Mountain 
fever,  although  they  have  many  points  in  common;  that  the 
Mexican  t>'phus  is  communicable  to  the  monkey;  and  that  it 
may  be  transmitted  by  an  insect  (Pediculus  vestamenti).  Some 
of  these  results  are  confirmatory  of  very  recent  results  obtained 
by  others,  but  on  April  23  they  were  able  to  announce  the 
discovery  of  a  micro-organism,  apparently  a  bacillus,  in  the  blood 
of  typhus  patients  and  in  the  insect.  It  seems  reasonable  to 
believe  that  this  organism  may  be  the  cause  of  typhus  fever. 

While  courageously  and  devotedly  pushing  this  and  other  work 
on  to  completion  Dr.  Ricketts  was  stricken  with  typhus,  and  the 


Howard  Taylor  Ricketts  7 

unfinished  investigations  of  such  fundamental  importance  must  be 
taken  up  by  others.  Thus  a  young  and  noble  career  of  great 
achievement  and  of  large  service  to  humanity  came  to  a  sudden 
and  heroic  end,  and  a  new  name  was  placed  on  the  martyr  roll  of 
science. 

Those  near  to  him  know  that  he  fully  understood  the  dangers 
to  which  he  would  be  exposed  and  the  risks  he  would  run.  He 
decided  he  would  take  those  risks,  meet  the  dangers  with  all  pos- 
sible means  of  prevention,  and  do  the  work  that  would  come  to  his 
hands.  And  so  he  made  the  great  sacrifice  and  gave  all  that  a  man 
can  give  for  his  fellow-men. 


CONTRIBUTIONS  TO  MEDICAL  SCIENCE  BY 
HOWARD  TAYLOR  RICKETTS 


A  NEW  MOULD-FUNGUS   AS   THE   CAUSE   OF  THREE 

CASES  OF  SO-CALLED  BLASTOMYCOSIS  OR 

OIDIOMYCOSIS  OF  THE  SKIN/ 

H.     T.     RiCKETTS, 
Fellow  in  Cutaneous  Pathology  in  Rush  Medical  College. 
(From  the  Pathological  Laboratory  of  Rush  Medical  College.) 

The  following  cases  of  oidiomycosis  (blastomycetic  dermatitis, 
Gilchrist)  of  the  skin  have  been  studied  recently,  and  a  mould- 
fungus  isolated  from  each. 

Case  I. — A  German,  73  years  old,  a  laborer,  and  of  negative 
family  history,  came  to  my  dispensary  clinic  at  Rush  Medical 
College  in  October,  1900. 

In  February,  1898,  a  pimple  appeared  on  the  scalp  above  the 
lobe  of  the  left  ear.  It  became  a  pustule  which  crusted,  increased 
in  size,  and  gradually  presented  a  verrucous  surface.  Extension 
occurred  with  some  rapidity,  the  oldest  portions  heahng.  When 
he  was  first  seen  nearly  the  whole  left  side  of  the  scalp  and  temporal 
region,  including  the  adjacent  skin  of  the  ear,  had  been  covered  by 
the  process.  Six  months  before  I  saw  him  another  pimple  appeared 
in  the  left  malar  skin,  which  now  measured  two  inches  in  diameter. 
The  center  appeared  flat,  depressed,  crusted,  and  bled  easily. 
The  peripheral  portion  was  elevated  from  a  half  to  one  centimeter 
above  the  skin  level,  and  was  covered  by  very  large  papillary 
growths.  The  surrounding  skin  of  both  lesions  was  infiltrated, 
reddened,  and  beset  with  many  minute  abscesses.  Healing  had 
occurred  over  a  large  part  of  the  left  scalp,  leaving  an  active 
verrucous  periphery.     There  is  no  history  of  venereal  disease. 

Verrucous  tissue,  and  pus  from  the  small  abscesses,  mounted 
in  potassium  hydrate  solution,  uniformly  show  an  organism  existing 
singly,  in  pairs,  or  in  small  groups,  the  members  of  which  are 
capsulated,  possess  a  granular  protoplasm  which  often  contains 
many  refractive  spherules  and  occasionally  vacuoles;  they  pro- 
liferate by  budding.     An  adventitious  capsule  is  found  surrounding 

'A  preliminary  report,  Jour.  Boston  Soc.  Med.  Sci.,  igoi,  5,  p.  453- 

II 


12  Contributions  to  Medical  Science 

many  cells.    This  examination  repeated  many  times  gives  similar 
findings. 

Tubes  of  ordinary  media  were  inoculated  several  times  from 
the  verrucous  tissue  and  from  the  abscesses  with  no  growth  except 
that  of  cocci  and  bacilli.  Eventually,  however,  a  maltose  agar, 
prepared  after  the  formula  of  Sabouraud  for  the  ringworm  fungi, 
was  inoculated  with  pus  from  the  abscesses  in  the  reddened  areola, 
the  skin  being  first  cleansed  with  alcohol.  After  six  days  small 
hyphal  colonies  were  noted  on  all  of  the  tubes.  They  increased  in 
size  slowly,  the  hyphae  reached  the  surface  of  the  tubes,  and  the 
substratum  presented  a  moulded  appearance.  Eventually  aerial 
hyphae  (Plate  i,  Fig.  i)  covered  the  whole  inner  surface  of  the 
tube.  Under  the  microscope  this  aerial  structure  consists  of  fine 
branching  threads  beset  with  lateral  pedunculated  conidia  which 
multiply  in  situ  by  a  budding  process.  That  portion  of  the  growth 
which  infiltrates  the  substratum  is  composed  of  similar  hyphae, 
which,  however,  are  larger  and  plainly  segmented  and  produce 
lateral,  sessile,  or  pedunculated  conidia  and  unicellular  offshoots. 
The  reverse  surface  of  agar  slants  exhibits  a  rich,  even,  golden- 
brown  color,  hke  that  of  a  well-colored  meerschaum.  Growth 
occurs  slowly  but  steadily  on  potato,  blood-serum,  gelatin,  and  in 
various  bouillons.  In  the  last-mentioned  medium  a  coherent, 
fluffy  mass  forms,  the  supernatant  fluid  remaining  clear.  The 
organism  does  not  ferment  saccharine  solutions. 

ProHferation  occurs  by  the  segmentation  of  hyphae  and  the 
ab junction  of  the  resulting  oval  cells;  by  the  formation  of  terminal 
and  lateral  spore  groups  or  conidia,  and  by  the  budding  of  ascus- 
like  cells. 

A  nvunber  of  subcutaneous  and  intraperitoneal  inoculations 
into  animals  were  without  result.  However,  a  dog  which  received 
an  intravenous  inoculation  died  in  one  month  from  a  mycosis  of 
the  entire  lung  tissue.  Cultures  of  the  organism  were  obtained 
from  the  nodules  comprising  the  consohdation.  Microscopically, 
the  organisms  as  they  existed  in  the  fresh  lung  consisted  of  spherical 
and  budding  cells  which  usually  were  more  or  less  filled  with  small 
structureless  refractive  spherules.  Similar  bodies  were  found 
free,  and  it  is  thought  that  transition  forms  have  been  observed 


A  New  Mould-Fungus  Causing  Oidiomycosis  of  Skin     13 

between  these  and  the  adult  capsulated  organism.  There  were  no 
hyphae  in  the  lung  tissue. 

A  portion  of  the  nodule  containing  spherical  organisms  was 
inoculated  into  a  hanging  drop  of  bouillon;  in  a  few  hours  the 
spherical  cells  sprouted  hyphae,  and  within  twenty-four  hours 
only  a  few  spherical  cells  were  found,  the  remainder  having  devel- 
oped one  or  more  hyphal  elements  (Plate  i,  Fig.  2),  In  cultures, 
also,  similar  spherical  cells  filled  with  the  refractive  bodies  spoken 
of  have  been  found  repeatedly. 

Histologically,  the  human  tissue  presents  two  or  three  interest- 
ing features.  The  verrucous  papillae  are  remarkably  large,  and 
many  give  off  secondary  branches.  There  is  an  unusual  amount 
of  abnormal  cornification  throughout  the  epidermis.  The  number 
of  miliary  abscesses  is  large,  and  it  has  been  possible  to  trace  the 
formation  of  multinuclear  giant  cells  from  the  cells  of  the  stratum 
mucosum.  A  unique  feature  in  the  cell  infiltrate  of  the  corium 
is  the  strikingly  large  number  of  eosinophiles.  These  are  found 
also  in  the  intra-epithelial  abscesses  and  in  interepithehal  spaces. 

Tubercle  bacilli  could  not  be  found. 

Case  II. — A  PoHsh  woman,  of  good  parentage,  and  with  no 
tuberculous  or  venereal  history.  The  disease  began  about  eighteen 
months  ago  as  a  pustule,  and  has  covered  the  right  side  of  the 
face,  the  bridge  of  the  nose,  and  has  extended  moderately  on  to 
the  supra-orbital  skin. 

When  first  seen  by  Dr.  Montgomery,  to  whom  I  am  indebted 
for  the  privilege  of  studying  the  case,  healing  had  occurred  for 
the  most  part,  except  at  the  periphery,  where  there  was  still  a 
verrucous  structure.  Located  here  and  there  in  the  periphery 
and  in  the  central  scar  tissue  were  a  number  of  small  projecting 
tubercles,  which  at  first  were  considered  pustules,  but  on  being 
incised  were  found  to  consist  of  a  cellular  or  mucoid  substance. 
Cultures  were  at  first  taken  from  the  verrucous  tissue,  but  the 
contamination  was  so  great  that  the  tubes  were  abandoned.  At 
this  time  an  examination  of  tissue  in  potassium  hydrate  solutions 
showed  the  presence  of  numerous  ''blastomycetes."  Subse- 
quently, several  of  the  myxoma-like  nodules  which  were  covered 
with  smooth  skin  were  cleansed  with  green  soap  and  alcohol,  the 


14  Contributions  to  Medical  Science 

overlying  epidermis  incised,  the  soft  contents  scraped  out  and 
inoculated  on  various  media.  It  was  demonstrated  at  this  time 
that  these  nodules  contained  organisms.  Two  weeks  later  the 
development  of  a  mould-fungus  was  noted  in  some  of  the  tubes. 
Growth  proceeded  slowly,  and  eventually  the  organism  in  its 
gross  and  microscopic  aspects  appears  identical  with  the  organism 
of  the  preceding  case.  Animal  experiments  have  not  yet  been 
made. 

The  tissue  removed  for  histological  study  included  a  myxoma- 
like  nodule  and  a  small  amount  of  verrucous  tissue.  The  organisms 
are  found  in  small  groups  in  the  corium.  There  are  a  number  of 
tubercles  resembling  closely  those  of  tuberculosis.  Tubercle  bacilli 
cannot  be  demonstrated.  As  in  the  preceding  case,  eosinophiles 
are  very  conspicuous  as  a  part  of  the  cell  infiltrate. 

Case  III. — (From  Professor  Senn's  surgical  clinic.  I  wish  to 
thank  Professor  Senn  for  permission  to  use  this  material.)  A 
German  woman,  33  years  old,  giving  no  personal  or  family  history 
of  tuberculosis,  venereal  disease,  or  carcinoma. 

Two  years  ago  three  pustules  appeared  simultaneously;  one 
on  the  left  cheek,  the  second  on  the  left  wrist,  and  the  third  on  the 
right  buttock.  They  increased  in  size,  became  verrucous,  and  the 
last  two  disappeared  spontaneously  after  about  three  months. 
That  on  the  face  progressed,  and  eventually  measured  about  two 
inches  in  diameter;  presented  a  depressed  center,  and  an  elevated 
periphery,  composed  of  very  coarse  papillary  processes;  the 
surrounding  areola  contained  many  minute  abscesses. 

The  diseased  tissue  was  removed  by  Dr.  Graham,  and  through 
accident  was  immersed  in  Zenker's  fluid,  for  thirty  or  forty  seconds. 
It  was  immediately  put  into  running  water,  and  washed  for  two 
hours.  It  was  feared  that  cultures  might  not  be  obtained  because 
of  this  treatment.  However,  a  portion  of  the  verrucous  tissue  was 
thoroughly  disintegrated  in  a  sterile  mortar,  the  resulting  pulp 
suspended  in  bouillon,  and  inoculations  made  on  all  available 
culture  media  and  agar  plates.  After  four  days  rosettes  of  hvphae 
were  seen  to  have  grown  out  from  a  number  of  small  fragments 
of  tissue  which  had  been  transplanted.     Growth  proceeded  as  in 


A  New  Mould-Fungus  Causing  Oidiomycosis  of  Skin     15 

the  two  preceding  cases,  and  eventually  the  same  mouldy  surface- 
appearance  resulted.  Microscopically,  this  organism  differs  in 
no  way  from  the  two  preceding. 

A  portion  of  the  tissue  was  inoculated  subcutaneously  in  the 
inguinal  region  of  a  guinea-pig.  After  a  week  a  nodule  developed, 
which  doubled  in  size  at  the  end  of  the  second  week,  and  was 
found  to  contain  cheesy  pus  which  on  cultures  yielded  the  organism 
isolated  from  the  human  tissue.  Spherical  capsulated  fungus 
cells  were  found  microscopically;  no  tubercle  bacilli  could  be 
demonstrated.  A  portion  of  this  pus  mounted  in  hanging  drop 
exhibited  a  growth  of  hyphae  from  the  spherical  cells,  many  of 
which  contained  the  refractive  bodies  alluded  to  above. 

Aside  from  the  usual  features  noted  in  "  blastomycetic  derma- 
titis," the  presence  of  large  numbers  of  infiltrating  eosinophils 
characterizes  this  case  as  it  does  the  two  preceding.  No  tubercle 
bacilH  could  be  demonstrated  in  the  human  tissue. 

The  organisms  so  far  described  in  connection  with  this  disease 
have  been  of  the  blastomyces  and  oidium  types.  Hence  it  is 
somewhat  startling  that  from  each  of  three  successive  cases  mould- 
fungi  were  cultivated  which  appear  identical. 

The  question  must  soon  be  decided  as  to  whether  these  varying 
organisms  obtained  from  a  constant  cHnical  entity  are  to  be  con- 
sidered of  common  generic  or  specific  dignity.  In  considering 
this  subject  recourse  must  be  had  to  the  observations  constantly 
made  by  botanists  that  the  members  of  a  particular  genus  or  even 
of  a  particular  species  are  subject  to  variations  in  morphology  and 
proHferation  which  may  often  be  remarkable.  It  is  to  be  noted, 
also,  that  many  botanists  consider  blastomyces-like  forms  and 
oidium-like  organisms  as  the  conidial  stages  of  somewhat  higher 
fungi.  In  view  of  these  facts  and  in  view  of  the  fact  that  the 
various  organisms  exist  as  the  cause  of  a  constant  clinical  entity, 
the  conclusion  is  reasonable  that  the  fungi  are  very  closely  related 
organisms.  This  is  substantiated  by  the  observation  that  the 
higher  types  (that  is,  the  mould-fungi)  possess  all  the  morpho- 
logical and  proUferative  possibihties  of  the  oidium-like  and  blasto- 
myces-like forms.    The  conclusion,  then,  seems  reasonable  that  the 


1 6  Contributions  to  Medical  Science 

latter  are  adaptation  forms  or  conidial  stages  of  the  mould-fungi 
which  have  been  considered  in  this  communication,  aerial  fructi- 
fication having  been  suppressed  in  the  simpler  forms. 

It  is  important  to  note  that  Ophuls  and  Moffitt,  in  June,  1900, 
pubHshed  an  account  of  a  mould-fungus  cultivated  from  the  so- 
called  protozoic  disease  first  described  by  Wernicke  and  studied 
in  detail  later  by  Rixford  and  Gilchrist.  All  previous  writers 
had  considered  the  organism  a  protozoon.  The  mould-fungus 
of  Ophuls  and  Moffitt,  however,  was  found  in  all  accessible  lesions 
and  a  disease  was  produced  in  animals  similar  to  that  in  the  patient, 
and  the  study  of  the  organism  from  the  lesions  in  hanging  drop 
cultures  proved  their  identity  with  the  mould-fungus  obtained  on 
solid  agar.  In  tissues  the  organism  existed  as  a  spherical  capsulated 
cell,  filled  with  refractive  globules  Hke  those  existing  in  the  organ- 
isms isolated  from  the  three  cases  presented  herewith. 

A  study  of  the  facts  at  hand  points  toward  the  identity  of  the 
mould-fungus  of  Ophuls  and  Moffitt  with  those  cultivated  from  the 
three  cases  of  oidiomycosis  of  the  skin  under  consideration. 

conclusions. 

1.  A  group  of  closely  related  organisms,  which,  however,  show 
rather  constant  differences,  exists  as  the  cause  of  blastomycetic 
dermatitis  (Gilchrist). 

2.  The  simpler  organisms  probably  are  adaptation  forms  or 
conidial  stages  of  the  more  complex  mould-fungus,  possessing, 
however,  fixed  specific  characteristics. 

3.  The  protozoic  (?)  disease  of  Posadas,  Wernicke,  Rixford, 
and  Gilchrist,  and  others  may  be  a  general  disease  of  which  the 
so-called  blastomycetic  dermatitis  is  a  local  manifestation. 

4.  Those  cases  of  oidiomycosis  of  the  skin  in  which  a  mould- 
fungus  exists  as  the  cause  seem  to  be  characterized  by  an  eosinoph- 
ilous  cell-infiltrate. 

(Since  the  above  was  written,  cultures  have  been  made  from 
two  additional  cases  of  "blastomycetic"  dermatitis,  and  a  mould- 
fungus,  appearing  identical  with  the  three  referred  to,  isolated 
from  each.) 


PLATE    1 


Fig.  1. 


^rm^is^mm^ 


■y^-^_ 


-^"X. 


/ 


'^ 


Fig.  2. 


A  New  Mould-Fungus  Causing  Oidiomycosis  of  Skin     17 

EXPLANATION  OF  PLATE  i. 

Fig.  I. —  Showing  aerial  hyphae  with  lateral  conidia,  and  terminal  pseudo- 
ascospores  (?).     (Sketched  from  a  culture  tube  under  a  low  power.) 

Fig.  2. —  The  development  of  hyphae  from  spherical  capsulated  spore-containing 
(?)  cells.  Fluid  from  a  mycotic  pulmonary  nodule  (dog)  containing  the  organisms 
was  mounted  in  a  bouillon  hanging  drop  and  the  growth  observed. 

The  nature  of  the  enclosed  spherules  is  uncertain.  A,  B,  C,  and  D  illustrate 
successive  stages  of  hyphal  growth;  E  and  F,  the  later  formation  of  pedunculated 
conidia.  The  larger  spherical  cells  were  10-13  niicrons  in  diameter;  the  conidia 
3-4  microns. 


OIDIOMYCOSIS  (BLASTOMYCOSIS)  OF  THE  SKIN  AND 

ITS  FUNGI/ 

Howard    T.    Ricketts, 
Fellow  in  Cutaneous  Pathology  in  Rush  Medical  College. 

From  the  Dermatological  Clinic  and  the  Pathological  Laboratory  of  Rush  Medical  College  in  affiliation  with 

the  University  of  Chicago. ) 

CONTENTS. 

Introduction 

I.  History  and  Pathogenic  Properties  of  Oidia 

Blastomycetes  and  Saccharomycetes  in  Human  and  Animal  Lesions 
Other  than  Blastomycetic  Dermatitis        .... 

Experimental  Blastomycelial  and  Similar  Infections  in  Animals 
Blastomycetic  Theory  of  Tumors   ...... 

Sporothrix  Schenckii     ........ 

II.  Summary  of  So-called  Blastomycetic  Infections  in  Man 

1.  Saccharomycosis  hominis,  Busse 

2.  Saccharomycosis  humaine,  Curtis 

3.  Blastomycetic  dermatitis,  Gilchrist 

4.  The  Protozoic  (?)  Infection  of  Wernicke  and  Others 

5.  Summary         ....... 

III.  Clinical,  Mycological,  and  Histological  Study  of  Twelve   New 
Cases  of  Oidiomycosis  of  the  Skin 
Case  I 
Case  II 
Case  III 
Case  IV 
Case  V 
Case  VI 
Case  VII 
Case  VIII 
Case  IX 
Case  X 
Case  XI 
Case  XII 

IV.  Analysis  of  Blastomycetic  Infection  of  the  Sb:in 
Clinical  Data        ....... 

Diagnosis  ....... 

Prognosis    ........ 

Treatment  ....... 


PAGE 
19 

19 

23 
26 

29 
30 

30 
30 

35 

36 

SI 
S6 

56 
S6 
72 
77 
87 
95 
96 

lOI 

no 

112 
116 
116 

1x8 

119 
119 
126 
128 
129 


•  From  Jour.  Med.  Research,  1901,  6,  p.  374. 
Path.  Soc,  1899-1901,  4,  p.  348. 


A  preliminary  summary  is  published  in  Trans.  Chic. 


Oidiomycosis  of  the  Skin  and  Its  Fungi 


19 


Pathogenesis 

Pathological  Anatomy 

Serum  Reactions  . 

Special  Histological  Features 

Comparative  Study  of  Organisms, 

Classification  and  Nomenclature 

General  Summary 

Conclusions 

V.  Bibliography   .... 

VI.  Explanation  of  Plates   . 


with  Table 


PAGE 

130 
132 
134 
^35 
144 

159 
162 

167 

168 
173 


INTRODUCTION. 

Since  attention  was  called  to  the  existence  of  blastomycetic 
dermatitis  of  Gilchrist  in  Chicago  and  vicinity  the  number  of  cases 
observed  has  rapidly  multiplied.  I  am  greatly  indebted  to  Pro- 
fessor Hyde  and  Professor  F.  H.  Montgomery  for  the  opportunity 
to  study  the  clinical  features  of  a  number  of  cases,  to  secure  material 
for  investigation  of  the  fungi  concerned  and  of  the  histological 
changes,  and  for  their  many  courtesies.  For  co-operation  and 
advice  I  wish  to  thank  Professor  Hektoen,  in  whose  laboratory 
the  work  has  been  done. 

The  organisms,  which  I  have  isolated  from  cases  of  blasto- 
mycetic dermatitis,  seem  to  fall  into  three  groups  which  have  so 
many  biological  and  morphological  features  in  common  that  they 
probably  represent  different  species  of  a  common  genus.  They 
all  belong  to  the  type  of  fungi  that  Link  designated  as  Oidium. 
This  being  the  case,  I  have  considered  it  advisable  to  briefly  review 
the  Hterature  in  regard  to  oidia,  more  particularly  the  pathogenic 
forms,  before  taking  up  the  study  of  the  so-called  blastomycetic 
infections  proper. 

I.  HISTORY  AND  PATHOGENIC  PROPERTIES  OF  OIDIA. 
Link  gave  the  name  Oidium  to  a  genus  of  the  fungi,  having  an 
elementary  organization,  many  species  living  as  moulds.  They 
have  more  or  less  ramifying  and  segmented  filaments,  which 
produce  at  their  extremities  short  cells,  very  often  rounded.  These 
cells,  which  are  united  end  to  end,  rosary-like,  detach  themselves 
when  mature.  These  are  the  spores,  which  germinate  and  produce 
filaments  like  those  from  which  they  came. 


20  Contributions  to  Medical  Science 

Of  the  oidia,  the  Oidium  albicans  was  the  first  of  medical  im- 
portance, and  began  to  occupy  the  attention  of  writers  in  1840. 
We  learn  from  Robin  that  Berg  first  saw  the  filaments  and  spores 
of  this  oidium  under  the  microscope  and  recognized  the  disease  it 
produced  in  the  mouth,  pharynx,  and  esophagus,  the  stomach  and 
rectum.  Gruby,  in  1842  and  1844,  published  reports  of  his  investi- 
gations, in  which  he  considered  the  parasite  of  thrush  analogous  to 
Sporotrichum.  He  used  the  name  ''Species  Sporotrichum  afl&nis, 
Gruby."  There  followed  a  discussion  between  Gruby  and  Berg 
as  to  the  nature  of  the  organism.  Oesterlen's  findings  were  similar 
to  those  of  Gruby.  Vogel's  work  was  in  the  nature  of  a  review; 
he  offered  the  opinion  that  the  fungus  did  not  grow  upon  the 
intact  mucous  membrane,  but  upon  the  exudation  into  which 
the  latter  is  converted  when  it  begins  to  decompose.  Between  the 
years  1841  and  1847  other  writers,  including  Eschericht,  Hannover, 
Hoenerkopf,  Baum,  Slayter,  Gubler,  and  Remak,  described  thrush 
clinically  or  from  a  mycological  standpoint.  Robin  followed  with 
his  classical  works  in  1847  ^-^^  i^SS-  He  observed  that  the  spores 
again  grow  into  the  tubular  form  of  the  organism,  a  matter  not 
understood  previously  and  in  reahty  not  thoroughly  established 
until  Grawitz  in  1880  pubHshed  an  account  of  his  investigations. 

Since  Robin's  time  much  has  been  written  concerning  the 
botanical  position  of  this  fungus,  and  the  non-pathogenic  Oidium 
lactis.  Instances  of  morbid  processes  caused  by  the  former,  aside 
from  thrush,  have  been  reported  from  time  to  time. 

In  1848  Bernard  expressed  the  conviction  that  yeast-like 
organisms  found  their  way  to  the  gall-bladder.  Reubold  in 
1854  confirmed  what  had  been  observed  in  regard  to  the  effects  of 
Oidium  albicans  upon  the  epithelium  of  the  mouth,  pharynx, 
esophagus,  and  vocal  chords.  He  also  found  the  fungus  in  the 
stomach,  intestines,  and  respiratory  passages,  where  it  had  not 
penetrated  the  epithelium.  Virchow  found  the  submucous  tissue 
of  the  esophagus  invaded  by  the  oidium.  In  i860  v.  Zenker  found 
multiple  brain  abscesses  in  a  man  affected  with  hemiplegia,  and 
a  coexistent  "Soor"  of  the  throat.  "  Soor-Massen  "  were  found  in 
the  abscesses.  (Baumgarten  holds  that  the  cerebral  abscesses 
must  have  been  due  to  a  mixed  infection  with  pyogenic  bacteria.) 


Oidiomycosis  of  the  Skin  and  Its  Fungi  21 

In  1869  Parrott  described  a  nodule  of  Oidium  albicans  in  the  lungs 
and  a  peritoneal  localization.  E.  Wagner  (1868)  found  oidium 
threads  in  the  connective  tissue  of  the  esophagus  and  in  neighboring 
blood-vessels.  A.  Vogel  found  similar  conditions.  Grohe,  in 
1870,  observed  mycelial  abscesses  in  the  Hver.  Slawjansky  found 
Oidium  albicans  in  a  spongy,  pea-sized  nodule  in  the  lung  of  a 
patient  who  had  died  of  pneumonia.  Ribbert  (1879)  described 
multiple  brain  abscesses  in  a  twelve-day-old  child  dying  with 
thrush  of  the  tonsils,  pharynx,  esophagus,  and  vocal  chords. 
Mycelium  was  found  in  one  abscess.  There  were  no  micrococci. 
Parrott  found  oidia  penetrating  the  tissue  of  the  esophagus  and 
stomach.  Birch-Hirschfeld  found  Oidium  albicans  in  the  lungs 
of  a  four-day-old  boy,  and  Rosenheim  in  a  case  of  fetid  bronchitis. 
Heller,  in  1888,  found  oidium  threads  penetrating  the  connective 
tissue  in  an  esophageal  ulcer.  Similar  conditions  existed  in  a  case 
of  laryngeal  thrush.  Schmorl,  in  1890,  obtained  an  oidium  from 
multiple  abscesses  of  the  kidneys.  The  patient  had  died  of  typhoid 
fever  and  had  extensive  thrush.  In  five  infants  dying  of  gastro- 
enteritis, Schmidt  found  thrush  deposits  in  the  walls  of  the  esopha- 
gus, larynx,  trachea,  and  larger  bronchi,  and  once  in  the  lung 
tissue.  The  conidial  form  predominated.  They  penetrated  the 
submucosa,  entering  the  blood-vessels,  and  in  two  cases  the  mus- 
cular layer  was  reached.  Ross  (1891)  found  Oidium  albicans  in 
cyst-hke  lesions  of  the  lungs.  Giulini  reported  a  case  of  ''Soor  der 
Vulva"  in  a  twenty-four-year-old  woman,  three  months  pregnant. 
A  few  easily  detached  islands  first  appeared,  and  in  a  few  days 
a  uniform  membrane  covered  the  vulva.  Guide  described  a  lung 
abscess  in  a  boy  three  years  old,  caused  by  Oidium  albicans.  Sud- 
den hemorrhage  resulted.  Preyhan  (1891)  discovered  spores  of 
the  aphtha  fungus  in  the  sputum  of  a  patient  suffering  from  pneu- 
monia and  hemorrhagic  pleuritis.  Brandenberg  (1893)  found 
Oidium  albicans  in  two  cases  of  angina,  and  Charrin  in  a  submaxil- 
lary abscess.  Langerhans  (1894),  at  the  autopsy  of  an  eighty- 
three-year-old  woman,  observed  round,  whitish  masses  filling 
the  uterus.  They  contained  mycelium  resembling  that  of  Oidium 
albicans.  No  cultures  were  made.  Grassett  (1893)  found  gingival 
abscess,  supposedly  caused  by  the  thrush  parasite,  but  staphylo- 


22  Contributions  to  Medical  Science 

cocci  were  present  in  large  numbers.  Heller  (1895)  examined 
post  mortem  the  mucous  membranes  of  twenty-five  cases  affected 
with  thrush.  In  some  cases  only  the  epithelium,  in  others  the 
underlying  tissue  was  invaded,  thromboses  being  found  in  six 
cases.  In  nine  cases  ulcers  were  present.  In  one  case  large  masses 
were  found  in  the  bronchi,  penetrating  the  blood-vessels.  He 
proved  experimentally  that  these  processes  were  not  due  to  post- 
mortem invasions.  V.  Herflf  (1895)  ^^  twenty-six  mycotic  infec- 
tions of  the  vagina  found  Oidium  albicans  in  sixteen,  Monilia 
Candida  in  four,  Leptothrix  vaginalis  in  one,  and  another  oidium- 
like  fungus  in  one.  Galli-Valerio  discovered  a  variety  of  Oidium 
albicans  in  the  discharges  of  a  child  dying  from  gastro-enteritis. 
It  formed  threads  and  chlamydospores,  and  produced  fermentation 
with  an  intense  odor.  He  concluded  that  oidia  may  be  a  cause  of 
gastro-enteritis  in  children.  The  organism  differed  in  cultural 
properties  and  virulence  from  t}^ical  Oidium  albicans  and  Oidium 
lactis.  Giuseppe  Cao,  considering  many  of  the  blastomycetes  so 
far  described  to  be  oidia,  experimented  with  forty-one  varieties 
isolated  from  sputum,  feces,  fruit-juice,  etc.,  and  found  many 
of  them  pathogenic  to  animals  in  varying  degrees.  He  divided 
them  into  four  groups,  depending  on  their  cultural  and  pathogenic 
properties. 

The  cultural  and  pathogenic  properties  of  the  Oidium  albicans 
and  other  closely  allied  oidia  have  been  studied  by  Grawitz,  Klem- 
perer,  and  many  others.  Grawitz  contended  that  it  was  not  an 
oidium,  but  a  budding  fungus  and  nearly  related  or  identical  with 
Mycoderma  vini.  Plaut  considered  it  identical  with  MoniKa 
Candida  (Bonorden),  a  widespread  lower  organism  belonging  to  the 
Torulaceae.  Others  have  considered  it  a  "blastomyces,"  or 
saccharomyces  (Hansen,  Jorgensen).  It  has  been  found  to  produce 
general  mycosis  in  rabbits,  guinea-pigs,  and  mice,  but  is  less 
pathogenic  to  dogs.  Roger  and  Charrin,  and  Otrowsky  were  able 
to  produce  a  high  degree  of  immunity  in  animals.  The  cultural 
properties  have  been  found  to  be  fairly  uniform,  but  certain  varia- 
tions in  morphology  and  biologic  properties  lead  to  the  conclusion 
that  there  are  different  varieties  or  adaptation  forms. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  23 

blastomycetes  and  saccharomycetes  in  human  and  animal 
lesions  other  than  blastomycetic  dermatitis. 

Organisms  somewhat  resembling  oidium  have  been  found  in 
various  conditions  in  man,  other  than  "  blastomycetic "  dermatitis 
and  tumors;   and  called  Blastomycetes  or  Saccharomycetes. 

Oudemans  and  Pekelharing  obtained  from  Pityriasis  capitis 
an  organism  which  they  called  Saccharomyces  capilitii.  They 
claimed  to  have  reproduced  the  disease  in  rabbits.  Buist  cultivated 
a  blastomyces  from  the  lesions  of  variola  and  vaccinia.  Babes 
found  blastomycetes  in  certain  ulcers  of  the  skin.  Colpe  described 
an  oval  yeast  as  the  only  organism  present  in  a  chronic  catarrh  of 
the  endometrium.  Busse  found  yeast-like  bodies  in  a  long-standing 
pyosalpinx;  also  in  the  products  of  a  three-months'  abortion. 
Cultures  were  sterile  in  both  cases.  De  Simoni  reports  that  in 
five  cases  of  hypertrophic  tonsils  he  found  blastomyces-like  bodies 
in  fresh  preparations  and  stained  tissue.  They  were  not  present 
in  normal  tonsils,  but  cultures  were  not  made.  It  is  not  clear  that 
these  bodies  were  not  the  result  of  degenerative  tissue  changes. 
Corselli  and  Frisco's  case  of  supposed  sarcoma  of  the  mesenteric 
lymph  glands  and  peritoneum,  with  metastases  in  the  thorax, 
seems  rather  to  have  been  an  inflammatory  process,  caused  by  a 
blastomyces.  The  organism  produced  similar  conditions  in  animals. 
Secchi  in  the  stained  sections  of  two  cases  of  keloid  acne  found 
blastomyces-like  bodies.  De  Stoecklin  examined  five  hundred 
cultures  from  suspected  diphtheritic  throats.  In  thirty-seven  were 
thrush-like  fungi,  which  he  considered  blastomycetes.  Nineteen 
of  these  cases  also  yielded  LoefB.er's  bacillus.  He  concluded  that 
the  fungus  intensified  the  pathogenic  effect  of  the  diphtheria 
bacillus,  and  that  the  fungus  alone  could  cause  a  severe  angina. 
Troisier  and  Achalme  found  in  a  case  of  thrush  an  organism  which 
they  considered  a  yeast  (a  saccharomyces).  It  differed  culturally 
from  the  Oidium  albicans.  Demme  (1891)  describes  a  red  budding 
fungus  (Saccharomyces  ruber)  as  a  contamination  of  milk.  Seven 
children  who  drank  infected  milk  suffered  from  gastritis.  The 
organism  was  not  pathogenic  for  guinea-pigs  and  dogs  when 
injected  subcutaneously,  but  when  infected  milk  was  fed,  gastro- 


24  Contributions  to  Medical  Science 

enteritis  resulted.  In  a  pseudo-membrane  of  secondary  syphilis 
of  the  throat  Teissier  found  a  pure  culture  of  a  yeast  resembling 
the  "levure  du  muguet."  He  concluded  that  the  syphilis  favored 
the  localization  of  the  yeast  infection.  Hannover  (1842)  found 
certain  torulae  in  fermenting  diabetic  urine,  and  De  Gaetano  the 
Saccharomyces  septicus  in  urinary  sediment.  Busse  obtained 
cultures  of  yeast  fungi  from  nasal  polypi  and  found  similar  bodies 
in  the  tissues.  Owing  to  contaminations,  he  was  unwilKng  to 
consider  the  organisms  the  cause  of  the  polypi.  Buschke  (1898) 
obtained  from  a  vaginal  secretion  a  yeast  which  produced  a 
cutaneous  blastomycosis  in  animals.  It  differed  from  the  organism 
described  by  Curtis  and  from  that  isolated  by  Maffucci  from  a 
skin  disease  in  a  guinea-pig.  Brazzola  (1896)  described  a  case 
of  general  infection  of  a  child  by  a  saccharomyces.  The  patient 
had  died  supposedly  of  diphtheria,  but  the  author  found  a  pure 
culture  of  a  saccharomyces  in  the  throat.  The  organism  resembled 
that  cultivated  by  Busse  from  a  case  of  pyemia,  and  caused  general 
infection  in  animals.  Buschke  also  isolated  blastomycetes  from 
five  out  of  six  cases  of  seborrhea.  One  organism  was  pathogenic 
to  animals.  Behla  (1897)  cultivated  blastomycetes  from  the 
so-called  Miescher's  tubules.  Previous  attempts  had  failed. 
Alexander  G.  R.  Foulerton  inoculated  animals  with  various  yeasts, 
including  one — Saccharomyces  tumefaciens  albus — which  he  isolated 
from  two  cases  of  pharyngitis.  Death  often  resulted  in  from  a  few 
days  to  three  or  more  weeks,  and  granulomatous  swelhngs  often 
developed  at  the  site  of  inoculation,  from  which  the  yeasts  were 
again  cultivated.  Stoewer  obtained  from  three  cases  of  keratitis 
pure  cultures  of  a  red  yeast,  which  proved  pathogenic  to  rabbits' 
eyes  when  injected.  He  also  produced  an  inflammatory  condition 
in  rabbits'  eyes  with  Curtis'  organism.  Lundsgaard  obtained  a 
pure  culture  of  a  blastomyces  from  a  case  of  hypopyon.  Inocula- 
tions caused  haziness  in  the  eyes  of  rabbits.  Busse 's  case  of 
blastomycetic  septicemia  and  Curtis'  of  myxoma-like  tumors  caused 
by  a  blastomyces  are  considered  later. 

Organisms  similar  to  the  above  have  been  found  in  diseases 
of  animals. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  25 

Monospora  bicuspidata,  a  blastomyces,  was  recognized  by 
Metschnikoff  in  a  disease  of  the  daphnia.  Its  morphology  and 
life  history  were  followed  out  histologically,  cultures  not  being 
obtained.  It  was  oblong,  often  tube-like  in  shape,  and  possessed 
a  needle-shaped  spore  lying  in  the  long  axis  of  the  cell.  The  organ- 
isms are  ingested,  the  capsule  dissolved,  the  spores  penetrate  the 
intestinal  wall  and  enter  the  circulation.  Lateral  conidia  sprout, 
and  these  by  a  budding  process  produce  adult  cells,  which  again 
form  spores. 

Pfeifer  found  torula  forms  in  bovine  vaccine  lymph.  Lymphangi- 
tis epizootica  (Lymfangite  epizootica,  Linfangite  farcinoide,  in  Italy; 
Farcin  de  riviere,  Farcin  d'Afrique,  in  France),  affecting  horses 
especially,  was  long  supposed  to  be  of  parasitic  origin.  Rivolta  called 
certain  highly  refractive  bodies,  which  were  present  constantly  in 
the  pus, "  Cryptococcus  farcinimosus  Rivoltae."  CanaHs  considered 
them  coccidia,  and  Plana  and  Galli-Valerio,  sporozoa.  The  disease 
affects  the  skin  and  upper  respiratory  passages  especially.  In 
the  skin,  nodules  and  ulcers  form;  glandular  metastases  occur, 
which  suppurate,  and  sinuses  form  in  the  subcutaneous  and  deeper 
muscular  tissue.  Recovery  usually  follows  in  a  few  months, 
although  in  some  cases  general  infection,  with  death,  occurs. 
Earlier  attempts  at  cultures  failed,  although  inoculations  with  the 
secretions  have  been  carried  out  successfully  by  Rivolta  and 
Micellone,  Bassi,  Chernier,  Chravat,  Delamotte,  Penpion,  and 
Boinet.  Aruch,  in  1892,  was  not  able  to  obtain  cultures,  but  in 
1895  Ferni  and  Aruch  cultivated  a  blastomyces  from  the  abscesses. 
In  unstained  preparations  of  the  pus,  budding  forms  and  empty 
capsules  were  found.  After  three  days  colonies  grew  on  potato. 
The  cells  were  rounded,  oval,  or  apiculate,  and  buds  formed  at  the 
ends.  Hyphae  are  not  mentioned.  Suppurating  nodules  were 
produced  in  the  testicles  of  rabbits  by  injecting  pure  cultures,  and 
the  organism  was  reclaimed  from  the  resulting  pus.  Tokishige  in 
Japan  and  Tartakowsky  in  Russia  obtained  similar  results. 
Tokishige's  organism,  however,  differed  markedly  from  that  of 
Fermi  and  Aruch.  From  thirty  to  fifty  days  were  required  for 
development  on  artificial  media,  and  in  time  the  surface  became 
folded  like  coils  of  intestines.     Microscopically,  hyphae  and  yeast- 


26  Contributions  to  Medical  Science 

like  cells  occurred  together.  Animal  experiments  gave  positive 
results.  Similar  epidemics  have  been  observed  in  Guadaloupe, 
France,  Algiers,  and  Norway. 

Ferran,  in  1889,  isolated  from  the  brain  of  mad  dogs  a  blastomy- 
ces  which  he  supposed  was  the  cause  of  the  mania.  Memmo  also 
found  an  organism  of  this  type  in  mad  animals.  Maffucci  and 
Sirleo  (1894)  gave  the  name  of  Saccharomyces  niger  to  a  blastomy- 
ces  cultivated  from  the  tissues  of  a  guinea-pig  which  died  in  a 
marasmic  condition.  The  animal  had  been  inoculated  with  a 
portion  of  the  Uver  of  an  embryo  guinea-pig,  the  mother  of  which 
had  died  of  tuberculosis.  The  lungs  and  l>Tnph  glands  contained 
myxoma-like  nodules,  and  similar  nodules  were  found  microscopi- 
cally in  the  kidneys.  They  contained  large  numbers  of  the  organism. 
There  was  great  hyperplasia  of  the  pulmonary  epithehum,  which 
was  at  first  considered  cancerous.  This  position  was,  however, 
abandoned  later.  Corselh  and  Frisco  (1895)  found  tumors  in 
the  lymph  glands  of  a  cachectic  guinea-pig,  with  metastases  in 
the  inner  organs,  and  isolated  a  blastomyces.  SanfeHce  (1895) 
designated  as  ''Saccharomyces  Hthogenes"  a  blastomyces  which 
he  cultivated  from  a  carcinomatous  metastasis  in  an  ox,  the  primary 
tumor  occurring  in  the  liver.  It  degenerated  into  calcareous-Kke 
masses  in  animal  tissue.  Also,  in  1897,  SanfeUce  pubHshed  experi- 
ments to  show  that  the  so-called  "  Taubenpocken "  is  caused  by  a 
blastomyces.  In  1898  Maffucci  and  Sirleo  again  cultivated  from 
a  guinea-pig  a  blastomyces  which  corresponded  to  their  first  one 
from  a  similar  source.  There  were  the  same  miliary  nodes.  San- 
feHce (1898)  called  an  organism  ''Saccharomyces  granulomatosus," 
which  he  obtained  from  a  granulomatous  nodule  of  a  pig.  Inocu- 
lation in  swine  produced  similar  lesions,  but  the  parasite  was  not 
pathogenic  to  other  animals. 

EXPERIMENTAL  BLASTOMYCELIAL  AND   SIMILAR  INFECTIONS   IN 

ANIMALS. 

Since  1848  infections  of  animals  with  yeasts  have  been  produced 
experimentally,  various  manifestations  resulting.  In  that  year 
Claude  Bernard  inoculated  dogs  intravenously  with  beer  yeasts 
to  determine  the  disposal  of  sugar.     The  animals  died.     Grohe 


Oidiomycosis  of  the  Skin  and  Its  Fungi  27 

(1869)  inoculated  yeasts  into  the  blood  and  peritoneal  cavity. 
Rapidly  fatal  mycosis  resulted,  nodules  containing  the  blastomy- 
cetes  being  found  in  various  organs.  Popoflf  (1872)  inoculated 
dogs  with  impure  yeasts  and  obtained  septicemic  and  typhoid 
conditions;  in  the  viscera  were  miliary  nodules,  in  which,  however, 
blastomycetes  or  myceHum  were  not  demonstrated.  Falk  (1896) 
failed  in  similar  experiments.  He  considered  Popofif's  results 
due  to  other  and  bacterial  organisms.  However,  Falk  may  have 
used  non-pathogenic  yeasts.  Neumayer  (1891)  found  that  animals 
could  be  fed  Hberally  with  yeasts  if  no  fermentable  substance  were 
introduced  at  the  same  time;  otherwise  gastro-enteritis  developed. 
No  pathological  changes  resulted  after  subcutaneous  inoculations. 
This  means  that  either  non-pathogenic  yeasts  or  non-susceptible 
animals  were  used.  Mosler  fed  animals  beer  yeasts;  dogs  were 
unaffected,  but  rabbits  developed  diarrhea.  Buist  produced 
pock-like  lesions  in  monkeys  using  beer  yeasts.  Roussy  produced 
fever  and  collapse  by  injecting  beer  yeasts  into  dogs  and  rabbits. 
He  isolated  a  ferment  from  cultures,  calling  it  pyretogenin.  A 
commission  later  decided  that  this  substance  was  invertin.  Raum 
(1891)  inoculated  rabbits  intravenously  with  various  yeasts, 
obtaining  dyspnea,  fever,  prostration,  and  death,  the  organisms 
often  being  recovered  from  the  blood  or  viscera.  A  thrush  culture 
also  proved  fatal,  the  fungus  being  found  in  liver  and  kidneys. 
Hueppe  (1892)  injected  wine  yeasts  into  guinea-pigs  intraperito- 
neally,  a  disease  resembHng  guinea-pig  cholera  resulting.  Sanfehce 
(1895)  cultivated  his  Saccharomyces  neoformans  from  fermenting 
fruit-juice.  In  guinea-pigs  small  nodules,  consisting  largely  of  the 
fungous  cells,  were  formed  in  all  organs  except  the  brain,  heart, 
and  suprarenals,  death  occurring  twenty  to  thirty  days  after 
inoculation.  The  nodules  to  him  suggested  neoplastic  growths. 
An  inoculation  into  the  breast  of  a  dog  resulted  in  a  condition 
which  he  called  carcinomatous.  There  were  widespread  metastases. 
He  emphasized  the  similarity  of  the  organisms  in  tissue  to  the 
so-called  coccidia  in  cancers.  Sanfelice's  Saccharomyces  Htho- 
genes  killed  white  mice  in  eight  days  after  subcutaneous  inocula- 
tion. Degenerative  masses  of  the  organism,  especially  in  sheep, 
took  up  calcium  salts.     General  infection  occurred  in  guinea-pigs, 


28  Contributions  to  Medical  Science 

and  the  blastomyces  Sanfelice  isolated  from  "  Gefliigelpocken " 
seems  also  to  have  been  pathogenic  to  animals.  The  Saccharomy- 
ces  niger  of  Maffucci  and  Sirleo  called  forth  enlargement  of  lymph 
glands  and  suppuration  at  the  point  of  inoculation  in  guinea-pigs, 
rabbits,  chickens,  and  dogs.  In  some  cases  mihary  and  submiliary 
nodules  occurred  in  the  lungs,  liver,  kidneys,  and  peritoneum, 
consisting  largely  of  epithelioid  cells  and  the  parasite.  Dogs  and 
guinea-pigs  were  about  equally  susceptible;  rabbits  were  less  so. 
Proliferation  of  the  epithelium  of  the  skin,  trachea,  and  pulmonary 
alveoli,  and  of  the  endothelium  of  the  lymph  channels  resulted. 
In  the  trachea,  in  one  case,  a  growth  developed  resembling  a  true 
papilloma.  However,  there  seems  to  be  no  reason  for  considering 
any  of  these  changes  other  than  inflammatory.  Of  great  impor- 
tance is  the  work  of  Lydia  Rabino witch,  who  among  forty  yeasts 
obtained  from  various  sources  found  only  eight  which  were  patho- 
genic to  animals  in  different  degrees.  Hence,  from  the  study  of 
a  single  yeast,  or  perhaps  more,  one  cannot  form  sweeping  con- 
clusions about  the  pathogenic  properties  of  yeasts  in  general. 
Casagrandi  (1897)  found  that  Saccharomyces  ruber  (Demme) 
was  pathogenic  for  guinea-pigs,  dogs,  and  mice  when  inoculated 
subcutaneously  or  into  the  abdomen.  The  Saccharomyces  septicus 
which  De  Gaetano  obtained  from  urinary  sediment  seems  to  have 
been  exceptionally  virulent,  producing  fatal  fibrinous  peritonitis 
and  septicemia  in  guinea-pigs  in  twelve  hours.  Noisette,  using 
a  commercial  yeast,  produced  a  mycosis  in  animals  similar  to  that 
which  is  obtained  with  the  Oidium  albicans.  The  effect  of  certain 
yeasts  in  rabbits'  eyes  observed  by  Stoewer  has  been  mentioned. 
Nesczadimenko,  using  two  spherical  and  one  oval  yeast,  inoculated 
mice,  rats,  rabbits,  guinea-pigs,  and  dogs  intraperitoneally,  fibri- 
nous and  purulent  peritonitis  resulting,  with  death  in  eight  to 
twelve  days.  Subcutaneous  injections  produced  inflammatory 
nodules,  which  suppurated  in  seven  to  nine  days.  Klein  isolated 
a  blastomyces  from  milk;  subcutaneous  inoculations  produced 
gelatinous  tumors  and  glandular  swelHngs  in  guinea-pigs.  Intra- 
peritoneal inoculations  caused  nodules  in  liver,  lungs,  pancreas, 
ovaries  or  testes,  and  intestines.  The  nodules  consisted  largely 
of  the  organism,  which  sometimes  produced  hyphal  forms  not 


Oidiomycosis  of  the  Skin  and  Its  Fungi  29 

noted  in  the  cultures.     The  lesions  are  similar  to  those  caused  by 
Curtis'  organism. 

Finally,  all  those  workers  who  claim  to  have  cultivated  blastomy- 
cetes  from  mahgnant  tumors  have  demonstrated  the  pathogenicity 
of  their  organisms  for  various  animals  in  greater  or  less  degree ;  and 
similar  organisms  concerned  in  blastomycetic  dermatitis  have  been 
proved  to  be  more  or  less  pathogenic  to  animals. 

BLASTOMYCETIC  THEORY  OF  TUMORS. 

Since  Virchow  described  certain  conditions  in  cancer-cells  as 
''endogenous  cell- formation,"  some  observers  have  held  that  these 
bodies  were  protozoa  (coccidia),  and  others  that  they  were  fungous 
bodies  (blastomycetes) ;  the  advocates  of  each  theory  claiming 
that  they  were  dealing  with  the  specific  cause  of  cancer.  Both 
theories  have  been  combated  by  many  pathologists,  who  hold 
that  the  bodies  in  question  are  a  form  of  degeneration  of  tissue 
cells.  This  matter  concerns  us  because  blastomycetes  are  held 
to  cause  malignant  tumors;  even  Sanfelice  affirms  that  hyaline 
bodies  and  blastomycetes  are  identical;  because  we  find  in  "blasto- 
mycetic" dermatitis  both  blastomycetes  and  hyaline  bodies,  the 
two  being  distinct  morphologically  in  staining  properties. 

Secchi  and  Anna  Stecksen  have  given  excellent  summaries 
of  the  Hterature  bearing  on  this  matter.  Leopold  also  has  recently 
added  original  observations. 

Pellagatti  concluded  that  the  blastomyces-like  bodies  which 
observers  have  noted  in  carcinoma  are  the  result  of  degeneration 
of  plasma  cells  into  hyaUn  spherules. 

As  noted  later,  the  bodies  which  Russell,  Ruffer  and  Walker, 
and  Plimmer  consider  the  parasites  of  malignant  tumors  are 
present  abundantly  in  oidiomycosis  of  the  skin. 

This  much  only  is  certain:  that  blastomycetes  which  are  able 
to  cause  septicemia,  pyemia,  and  local  morbid  conditions  in  animals, 
with  connective-tissue,  endothehal,  and  epithelial  hyperplasia, 
have  been  isolated  from  malignant  tumors.  The  evidence  is  not 
sufl&cient  to  prove  that  the  changes  produced  in  animals  are  other 
than  inflammatory. 


30  Contributions  to  Medical  Science 

sporothrix  schenckii. 

The  sporotrichal  infection  of  Schenck,  also  studied  by  Hektoen, 
is  of  interest  here,  because  of  certain  similarities  between  its  organ- 
ism and  some  of  those  causing  blastomycetic  dermatitis.  A  com- 
parison of  Fig.  7,  Plate  3,  of  Hektoen's  article  {Jour.  Exp.  Med., 
1900,  5),  with  our  Fig.  13,  Plate  7,  showing  a  form  of  the  organ- 
ism from  Case  I,  shows  considerable  similarity  between  the  two 
organisms. 

The  surface  appearance  of  the  sporotrichum  on  agar  also  is  simi- 
lar to  that  of  the  organism  from  Case  III.  The  pigment  production 
of  the  former  and  the  nature  of  the  morbid  process  caused  by 
the  fungus  seem  to  indicate  that  Sporothrix  Schenckii  occupies  a 
somewhat  remote  position  in  relation  to  the  organisms  of  oidiomy- 
cosis of  the  skin. 

The  trichophyton  fungi,  likewise,  in  their  morphology  and 
methods  of  reproduction  bear  considerable  resemblance  to  some 
of  the  oidia  under  consideration  (i.e.,  the  mould-fungi). 

The  determination  of  the  exact  relationships  of  these  various 
organisms  will  need  further  study. 

For  the  sake  of  clearness,  and  to  group  the  facts,  we  shall 
consider  in  some  detail  the  authentic  cases  of  infections  in  man  by 
oidium-hke  and  similar  organisms  aside  from  those  mentioned 
in  the  foregoing;  these  have  been  pubhshed  since  1894. 

II.     SUMMARY    OF  THE    SO-CALLED    BLASTOMYCETIC    INFECTIONS 

IN  MAN.' 

I.     SACCHAROMYCOSIS   HOMINIS,   BUSSE. 

Busse  in  1894  pubUshed  his  first  account  of  a  case  of  pyemia 
caused  by  a  pathogenic  yeast.  Three  further  communications 
appeared  during  the  three  following  years.  Also  Buschke  has 
furnished  contributions  based  on  this  case  and  its  organism,  with 
particular  reference  to  the  skin  lesions. 

'  The  terminology  used  by  the  various  authors  is  retained  in  the  following  summaries. 
Only  peculiar  cultural  features  are  included  in  the  abstracts;   details  are  to  be  found  in  the  table 
on  p.  148. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  31 

The  patient  was  a  woman,  thirty-one  years  old,  and  the  wife  of 
a  shoemaker.  Busse  and  Buschke  differ  as  to  the  time  of  appear- 
ance of  the  skin  lesions,  the  former  stating  that  while  a  patient  in 
the  clinic  a  number  of  small  round  ulcers  appeared  on  the  face; 
while  Buschke  insists  that  previous  to  the  trouble  in  the  leg  small 
ulcers  would  appear  on  the  neck  and  forehead,  which  sometimes 
healed  spontaneously.  Thus  the  latter  would  make  the  skin  the 
infection  atrium.  Besides  the  tibial  lesion,  all  accessible  lymphatic 
glands  were  found  to  be  enlarged.  CHnically,  the  tumor  below  the 
knee  resembled  a  gumma  or  a  softened  sarcoma.  From  it  forty  or 
fifty  cubic  centimeters  of  glairy,  reddish  fluid,  containing  macro- 
scopic granules,  were  evacuated.  From  this  pus  Busse  obtained 
impure  cultures  of  a  supposed  yeast  fungus.  Later  the  disease 
extended  to  the  knee-joint,  the  surrounding  tissues  became  swollen, 
the  joint  less  and  less  movable,  and  the  leg  flexed.  Busse  describes 
the  ulcers  on  the  face  as  small  and  round,  with  elevated  borders, 
and  secreting  glairy,  sticky  pus.  The  influence  of  neighboring 
foci  caused  larger  irregular  ulcers.  According  to  Buschke,  the 
primary  facial  efflorescences  resembled  acne  infiltrations,  the  apices 
of  which  underwent  necrosis.  Growth  and  coalescence  of  neighbor- 
ing lesions  eventually  formed  sensitive,  crater-Hke  ulcers,  which 
were  rather  broad,  but  also  involved  the  tela  subcutanea.  The 
borders  were  sharp,  irregular,  somewhat  undermined  or  precipitous, 
moderately  infiltrated,  and  surrounded  by  a  livid  zone.  The  bases 
were  of  soft  granulation  tissue  and  discharged  a  tenacious,  trans- 
lucent, grayish  or  brownish-red  secretion,  which  contained  coarse 
granules.  Subcutaneous  foci,  which  ruptured  through  the  skin, 
caused  similar  ulcers.  They  occasionally  began  in  hair  follicles. 
From  these  skin  lesions  both  Busse  and  Buschke  isolated  the 
organism  found  in  the  tibial  abscess.  A  beginning  ulcer  covered 
by  the  primary  crust  always  gave  a  pure  culture  of  the  fungus. 

In  a  few  months  there  formed  an  abscess  of  the  right  ulna, 
near  the  elbow,  and  another  of  the  left  sixth  rib.  From  the  former, 
pure  cultures  of  the  fungus  were  again  obtained.  The  latter 
abscess  was  not  incised.  The  fungus  was  not  found  in  the  urine, 
but  eight  weeks  before  the  patient's  death  Buschke  obtained  a  pure 
culture  from  the  left  median  vein. 


32  Contributions  to  Medical  Science 

The  patient  died  thirteen  months  after  the  appearance  of  the 
tibial  abscess  and,  according  to  Buschke,  a  year  and  a  half  after 
the  development  of  the  first  skin  lesions.  Preceding  death  there  were 
extreme  marasmus,  an  irregular  pulse,  and  a  variable  temperature. 

During  the  patient's  stay  in  the  clinic  the  tibial  abscess  was 
dissected  out  and  sent  to  Busse  for  examination.  The  pus  consisted 
of  ordinary  pus  cells,  large  numbers  of  giant  cells,  connective- 
tissue  cells,  and  the  organism.  The  giant  cells  were  so  numerous  that 
a  softened  giant  cell  sarcoma  was  suggested.  The  organisms  were 
present  in  enormous  numbers,  both  extracellular  and  intracellular, 
in  the  latter  case  occurring  both  in  giant  cells  and  mononuclear 
smaller  ceUs.  The  abscess  wall  consisted  of  an  outer  osseous  struc- 
ture, a  middle  zone  of  fibrous  tissue,  a  small  cell  infiltrate,  and  an 
inner  necrotic  zone.  The  organisms  were  found  in  large  numbers  in 
the  inner  zone  and  to  a  less  degree  in  the  middle  zone.  As  stated 
above,  the  fungus  was  obtained  in  a  pure  state  from  the  pus.  A 
noteworthy  characteristic  of  the  organism  was  the  formation  of  an 
adventitious  or  accidental  capsule,  also  described  by  Buschke,  and 
later  by  Curtis  in  connection  with  his  organism.  Internal  to  the 
adventitious  capsule  was  the  cell  membrane  proper,  which  was  a 
translucent,  homogeneous,  doubly  contoured  structure.  The  proto- 
plasm contained  many  moving  granules  and  some  more  highly 
refractive  bodies,  which  were  considered  fat.  Prohferation  in  the 
tissue  and  pus  was  by  budding,  no  mycelium  being  found.  The 
cells  were  either  spherical  or  oval.  Some  biscuit-shaped  figures 
and  sickle-like  bodies  were  also  found.  These  were  considered 
degenerating  forms. 

Buschke  studied  the  skin  lesions  in  detail.  The  tissue  of  the 
large  ulcer  on  the  forehead  and  the  t>'pical  focus  induced  by  experi- 
mental inoculation  of  the  patient  were  used.  In  the  necrotic  base 
of  the  ulcers  there  was  no  epithelium.  The  surface  of  the  tissue 
immediately  beneath  it  contained  large  numbers  of  parasites, 
some  bearing  the  accidental  membrane,  and  others  being  sickle- 
shaped.  Here  also  were  polynuclear  leukocytes,  red  blood  cells, 
and  fibrin.  The  number  of  parasites  decreased  toward  the  under- 
lying tissue.  In  what  he  termed  the  middle  zone  were  many  giant 
cells,  containing  from  six  to  twenty  peripherally  disposed  epitheHoid 


Oidiomycosis  of  the  Skin  and  Its  Fungi  33 

nuclei,  leukocytes,  and  the  fungous  cells  often  in  the  budding  state 
and  surrounded  by  the  accidental  capsule.  Abscesses  were  found 
in  the  papillary  layer  and  deeper  cutis,  containing  many  poly- 
nuclear  and  a  few  mononuclear  leukocytes  and  epithelioid  cells,  red 
blood  cells,  fibrin,  nuclear  detritus,  and  occasional  giant  cells. 
Parasites  occurred  freely  and  diffusely  in  this  zone.  The  elastic 
fibers  had  disappeared,  although  in  the  papillae  and  deepest  inflam- 
matory zone  traces  were  found.  Plasma  cells  occurred  here  and 
there  without  any  special  grouping.  The  deeply  lying  giant  cells 
had  fewer  nuclei  than  the  superficial,  and  cells  of  this  type  contained 
no  organisms.  The  epithelium  was  affected  in  two  ways;  either  it 
was  totally  destroyed,  as  in  the  base  of  the  ulcer,  or  it  underwent  a 
carcinoma-like  proKferation.  The  latter  occurred  particularly  at  the 
margin  of  the  ulcer.  Fungous  cells  were  found  in  the  horny  layer 
and  in  larger  numbers  in  the  deeper  rete  and  the  enlarged  pegs. 
They  occurred  within  epithehal  cells,  the  nuclei  of  which  were 
flattened  against  the  cell  periphery.  In  all  cases  where  the  yeast 
cells  came  in  relation  to  the  epithelium  the  latter  was  destroyed. 
No  mention  is  made  of  intra-epithelial  abscesses.  He  considered 
that  the  parasite  gained  entrance  into  the  epithelium  by  the  agency 
of  leukocytes.  The  number  of  organisms  in  the  epidermis  was  small 
compared  to  those  seen  in  the  cutis.  Buschke  considered  the  cutis 
to  be  involved  primarily,  the  epithelium  secondarily. 

The  necropsy  disclosed,  besides  the  tibial  lesion,  similar  abscesses 
in  the  right  ulna  and  the  left  sixth  rib,  and  granulation  foci  or 
abscesses  containing  slightly  cheesy  pus  in  the  lung,  left  kidney,  and 
spleen.  The  same  yeast  was  demonstrated  in  all  these  lesions. 
Many  microscopic  tubercles  occurred  in  the  lungs  without  the 
presence  of  tubercle  bacilH.  The  kidney  lesions,  varying  greatly 
in  size,  consisted  almost  entirely  of  yeast  cells,  which  furthermore 
infiltrated  the  kidney  in  a  diffuse  manner.  The  masses  in  the  spleen 
were  similar  to  those  in  the  kidney. 

The  cultures,  which  were  obtained  in  a  pure  state  from  the 
abscesses  in  the  ulna  and  the  rib  and  from  certain  skin  lesions, 
developed  freely  in  forty-eight  hours.  The  points  of  peculiar 
interest  are  that  it  proliferates  in  cultures  chiefly  by  budding; 
however,  elongated  cells  were  common,  and  Buschke  speaks  posi- 


34  Contributions  to  Medical  Science 

tively  of  mycelial  formation  with  attached  conidia.  The  organism 
produced  alcohol  and  carbon  dioxide  in  grape  sugar  solutions,  and 
gelatin  was  not  Hquefied.  The  formation  of  acid  was  not  mentioned. 
The  cells  on  culture  media  were  of  the  naked  type  usually,  but 
eventually  Busse  discovered  on  old  cultures  cells  with  the  accidental 
capsule.  Malt  extract,  bouillon,  gelatin,  plum-decoction  gelatin, 
and  glycerin  agar  with  malt  extract  constituted  the  most  favorable 
media.  No  top  growth  occurred  in  liquid  media,  and  in  old  cultures 
a  brown  pigmentation  was  acquired.  The  formation  of  endospores 
could  not  be  induced  by  the  use  of  high  temperatures  (41°  C.)  on 
a  moist  gj-psum  block  or  in  distilled  water.  The  use  of  H.  Moeller's 
method  led  to  demonstration  of  a  nucleus-Hke  structure.  On 
account  of  a  reaction  obtained  with  osmic  acid,  Busse  considered 
certain  refractive  globules  to  be  fat.  Pus  from  the  tissues  of  the 
patient  and  pure  cultures  of  the  organism  were  inoculated  into  the 
tibia  and  beneath  the  periosteum  in  dogs.  Chronic  inflammation 
resulted,  in  which  the  fungus  multipHed  greatly,  but  spontaneous 
recovery  occurred.  An  intraperitoneal  inoculation  of  pus  pro- 
duced a  fibrinous  peritonitis,  in  which  the  organism  was  found  in 
large  numbers.  Subcutaneous  inoculation  with  bouillon  cultures 
produced  inflammatory  thickenings,  which  sometimes  suppurated, 
but  eventually  healed  spontaneously.  Intravenous  inoculation  in 
dogs  and  rabbits  produced  no  general  disease.  White  mice  inocu- 
lated in  the  muscles  died  at  first  in  four  to  ten  days.  At  a  later 
period,  presumably  on  account  of  weakened  \drulence  of  the  organ- 
ism, the  mice  lived  from  two  weeks  to  thirty-three  days.  There 
were  local  inflammatory  changes  and  numerous  yeast  cells  in  the 
blood  and  vessels  of  the  lungs  and  kidneys,  the  glomeruli  of  the 
latter  being  chiefly  involved.  There  was  little  visceral  inflam- 
matory reaction.  On  the  whole,  dogs  were  not  very  susceptible. 
Rabbits,  guinea-pigs,  and  cats  reacted  similarly.  It  was  empha- 
sized that  fatty  degeneration  occurred  in  the  muscles  and  other 
organs  near  the  points  of  inoculation.  MyceHum  did  not  develop 
in  the  animal  tissues,  proUferation  occurring  by  the  usual  budding 
process.  Busse  attempted  to  produce  epithelial  proliferation  by 
inoculation  into  the  mammary  glands  and  testes,  but  purely  inflam- 
matory thickenings  resulted.     He  considered  the  disease  purely 


Oidiomycosis  of  the  Skin  and  Its  Fungi  35 

of  inflammatory  nature,  without  relation  to  sarcoma,  as  held  by 
Sanfelice. 

2,    SACCHAROMYCOSIS   HUMAINE,   CURTIS. 

Curtis  pubHshed  in  1896  a  complete  study  of  a  case  in  which 
myxoma-like  tumors  were  caused  by  a  yeast-like  organism.  A 
preliminary  report  had  already  been  published.  In  July,  1895,  ^ 
tumor  of  this  sort  came  into.  Curtis'  hands  in  a  fresh  condition.  It 
had  been  situated  on  the  right  thigh,  near  Scarpa's  triangle,  was  as 
large  as  two  fists,  and  clinically  seemed  a  deep  abscess  or  a  softened 
sarcoma.  Similar  lesions  were  situated  in  the  lumbar  region,  and 
near  the  right  ribs.  The  abscesses  contained  thick  and  flaky  pus. 
The  patient  had  been  in  good  general  health,  and  gave  no  history  of 
hereditary  disease.  Later  he  died  of  meningitis  of  undetermined 
nature.  Histologically  the  tumor  consisted  of  a  mucous  or  gelati- 
nous substance,  suggestive  of  myxo-sarcoma.  This  was  caused 
by  the  large  numbers  of  the  organism,  which  in  places  constituted 
almost  the  entire  tumor  substance.  In  the  center  was  a  network  of 
connective-tissue  fibers,  infiltrated  with  leukocytes,  the  yeast 
cells  lying  in  the  fiber  meshes.  A  zone  of  cell  infiltration,  and 
another  of  fibrous  lamellar  tissue  constituted  the  abscess  wall. 
The  parasites  were  intracellular  or  extracellular.  The  latter 
often  possessed  the  adventitious  capsule  described  by  Busse,  but 
intracellular  organisms  would  lose  this.  It  grew  best  on  acid  or 
neutral  media,  development  occurring  in  forty-eight  hours  to  three 
days  as  small,  opaque,  white  colonies,  which  fused  slowly.  In 
about  eight  days  cultures  acquired  a  fleshy  or  paste-like  consistence. 
Gelatin  was  not  liquefied.  Saccharose  solutions  were  fermented, 
with  production  of  alcohol  and  acetic  acid.  An  extraneous  capsule 
also  formed  on  cultures,  and  a  brownish  pigmentation  was  acquired 
eventually. 

Guinea-pigs  were  not  susceptible,  according  to  Curtis.  Anna 
Stecksen,  however,  produced  abscesses  and  septicemia  in  these 
animals  with  a  culture  which  Curtis  sent  her.  Subcutaneous  inocu- 
lations into  rabbits  caused  abscesses,  which  healed  spontaneously. 
A  general  disease  could  not  be  produced  by  intravenous  inoculations. 
In  white  rats  tumors  resulted,  which  resembled  those  seen  in  the 


36  Contributions  to  Medical  Science 

patient.  One  rat  died  after  three  months,  with  soUdified  lungs  and 
miUary  tumors  of  the  pleurae,  spleen,  and  kidneys.  Pure  cultures 
were  obtained  from  these  lesions.  Gray  mice  died  in  about  a 
month.  White  mice  developed  tumors  like  white  rats.  Dogs 
presented  variable  susceptibility,  small  nodules  or  ulcerating  infil- 
trations resulting.  Since  in  animals  the  tumors  exactly  resembled 
those  of  the  man,  the  conclusion  was  positive  that  the  yeast  caused 
the  human  disease.  Spores  and  nuclei  were  not  demonstrated 
clearly  in  this  organism.  The  fungus  received  the  name  of  Sac- 
charomyces  subcutaneus  tumefaciens. 

3.    BLASTOMYCETIC  DERMATITIS,    GILCHRIST. 

To  Gilchrist  belongs  the  credit  of  having  first  observed  and 
described  a  pure  skin  disease  caused  by  a  yeast-like  fungus.  The 
above  name,  which  he  applied,  has  been  used  by  all  subsequent 
writers  in  describing  this  condition.  So  far  it  is  exclusively  an 
American  disease,  the  skin  lesions  described  by  Buschke  being 
merely  concomitant  foci  in  the  pyemia  of  Busse's  case. 

I.  In  May,  1894,  at  a  meeting  of  the  American  Dermatological 
Association,  six  months  previous  to  the  appearance  of  Busse's 
first  article,  Gilchrist  demonstrated  sections  of  a  pecuHar  skin 
disease.  Dr.  Duhring  had  described  the  case  as  a  typical  scrofulo- 
derma on  the  back  of  a  man's  hand.  Tissue  was  excised  and  sent 
to  Gilchrist  for  examination.  Under  surgical  treatment  the  dis- 
ease was  cured  and  cultural  experiments  became  impossible.  The 
surface  of  the  sections  was  irregularly  papillomatous  and  covered 
with  granular  and  cellular  detritus.  The  homy  layer  was  pro- 
nounced, and  infiltrated  with  collections  of  leukocytes.  "The 
epithelium  was  enormously  hyper  trophied,  and  consisted  of  irregu- 
lar prolongations  of  various  shapes  and  sizes,  extending  downward 
into  the  corium.  The  most  noticeable  feature  in  the  rete  was  the 
presence  of  numerous  well-defined  miliary  abscesses  of  different 
sizes.  The  smallest  of  these  were  about  equal  in  area  to  that  of 
three  adjoining  epithehal  cells,  while  the  largest  were  just  visible 
to  the  naked  eye.  They  contained  polynuclear  leukocytes,  nuclear 
detritus,  and  a  few  detached  epithehal  cells,  now  and  then  a  giant 
cell,  a  number  of  pecuhar  nuclear  cells,  and  the  organism  which 


Oidiomycosis  of  the  Skin  and  Its  Fungi  37 

characterized  the  disease."  The  abscess  walls  were  made  up  of 
flattened  epithehal  cells.  Even  small  aggregations  of  polynuclear 
leukocytes  contained  one  or  more  organisms.  There  was  also  a 
diffuse  infiltration  of  the  rete,  with  leukocytes,  and  in  places  "the 
line  of  demarkation  between  the  epithelium  and  corium  was  not 
easily  recognized."  There  were  numerous  examples  of  premature 
cornification.  Pigment  was  absent  entirely  from  the  epithelium. 
The  papillae  were  greatly  deformed  and  nearly  reached  the  surface 
in  places.  Miliary  abscesses,  a  few  giant  cells,  occasional  mast-cells, 
a  large  amount  of  granulation  tissue  and  new-formed  blood-vessels, 
surrounded  by  leukocytic  and  endothelial  infiltration,  small  hemor- 
rhages, and  fibrin  constituted  the  chief  subcutaneous  lesions. 
Occasionally  a  myxomatous  appearance  occurred  near  the  surface. 
From  four  to  fifteen  organisms  were  found  in  the  miliary  abscesses, 
very  few  in  giant  cells,  and  occasional  examples  in  the  granulation 
tissue.  They  measured  from  ten  to  twenty-three  microns  in  diame- 
ter, and  were  spherical  or  oblong.  Structure:  First,  a  doubly 
contoured  capsule;  second,  a  light  peripheral  zone;  third,  a  central 
protoplasm;  and  fourth,  a  vacuole.  The  capsule  measured  from 
0.6  to  0.8  micromilHmeters  in  thickness.  The  peripheral  zone 
was  prominent,  of  varying  thickness,  and  stained  a  homogeneous 
reddish- white  with  carbol-fuchsin  and  safranin.  The  protoplasm 
was  seven  or  eight  microns  in  diameter,  reticular,  and  finely  granu- 
lar, and  possessed  in  addition  numbers  of  larger  granules  of  vary- 
ing sizes.  The  vacuole  appeared  structureless.  Multiplication 
took  place  by  budding.  The  capsule  became  thin  at  one  point 
and  the  bud  or  protoplasm  pushed  out.  The  capsule  of  the  bud 
gradually  became  thicker,  its  protoplasm  increased  in  size,  a  clear 
space  soon  separated  it  from  the  mother  protoplasm,  and  eventually 
the  capsule  united  between  the  two  portions.  The  parent  cell 
would  sometimes  produce  more  than  one  bud  at  the  same  time. 
Degeneration  forms  were  represented  by  sickle-shaped  bodies  and 
capsules,  with  no  central  staining  substance.  "The  opinion  was 
expressed  by  Gilchrist  before  the  society  that  these  organisms  should 
be  classed  as  belonging  more  to  plant  than  to  animal  fife.  It  was 
not  until  Busse's  report  appeared  that  these  organisms  were  posi- 
tively identified  as  blastomycetes." 


38  Contributions  to  Medical  Science 

2.  A  preliminary  report  of  this  case  by  Gilchrist  and  Stokes 
appeared  in  July,  1896,  and  an  extended  study  was  published  in 
1898. 

The  patient  was  a  man  thirty-three  years  old,  in  good  general 
health,  and  with  a  negative  family  history.  The  disease  began 
eleven  years  pre\"iously  as  a  pimple  behind  the  left  mastoid  process. 
This  ulcerated,  and  the  lesion  extended  forward,  encircling  the  ear 
and  spreading  to  the  left  side  of  the  face  and  chin,  affecting  the 
palpebral  and  supra-orbital  skin,  and  eventually  crossed  the  nose 
to  the  right  side  of  the  face,  forming  lesions  almost  symmetrical 
with  those  on  the  left  side.  The  disease  was  transplanted  to 
various  parts  of  the  body  during  the  eleven  years. 

Histologically  the  epithelial  hyperplasia,  intra-epithehal  abscess 
formations,  generaHzed  infiltration  of  the  epithehum,  with  poly- 
nuclear  leukocytes,  abscess  formation  in  and  infiltration  of  the 
cutis,  together  with  the  organisms,  showed  the  case  to  be  identical 
with  that  first  reported  by  Gilchrist.  There  was  much  granulation 
tissue  and  a  few  giant  cells  in  the  corium.  The  organism  was 
found  in  all  miliary  abscesses,  occasionally  in  the  granulation  tissue 
of  the  cutis,  and  to  a  small  degree  in  the  giant  cells  of  the  cutis. 
They  were  described  as  "doubly  contoured,  refractive,  round  and 
oval  bodies,  varying  in  size  from  ten  to  twenty  microns  .... 
presenting  buds  of  various  sizes.  A  vacuole  was  often,  although 
not  always,  discernible.  The  bodies  were  usually  found  singly  or 
in  pairs,  but  they  were  sometimes  arranged  in  groups."  Parasites 
occurred  occasionally  in  giant  cells,  but  not  in  mononuclear  cells. 
"Pseudo- tubercles"  in  the  corium  had  almost  the  typical  structure 
of  those  seen  in  tuberculosis.  It  is  stated  that  ''pseudo-tubercles 
were  more  numerous  in  the  papillomatous  or  chronic  variety  of  the 
lesions;  whereas,  in  the  acute  or  ulcerative  form,  miliary  abscesses, 
and  consequently  more  parasites,  were  present."  The  structure  of 
the  organism  was  identical  with  that  of  Gilchrist's  first  case. 
Occasionally  the  endosporium  was  extruded  through  a  break  in 
the  capsule;  a  second  body  would  sometimes  form  before  the  first 
was  detached;  occasionally  an  organism  possessed  a  "fibrous-like" 
structure  external  to  the  capsule;  a  half-moon  shaped  contraction 
of  the  protoplasm  was  seen  occasionally.    With  Unna's  polychrome 


Oidiomycosis  of  the  Skin  and  Its  Fungi  39 

methylene  blue  method  certain  coarse  granules  in  the  fungus 
protoplasm  stained  red. 

Cultures  from  the  tissues  were  greatly  contaminated  with 
pyogenic  cocci.  From  the  pus,  however,  pure  cultures  of  the 
fungus  appeared  in  one  week.  The  length  of  time  required  for 
development  is  noteworthy.  The  organism  was  capable  of  pro- 
liferation either  by  a  budding  process  or  by  the  intervention  of  a 
mycelium,  which  formed  lateral  and  terminal  conidia.  The 
extraneous  capsule  did  not  appear  in  cultures.  Certain  proto- 
plasmic granules  exhibited  Brownian  movement,  stained  deeply 
in  aniUne  dyes,  and  by  Millon's  reagent  appeared  to  be  albuminous. 
The  protoplasm  was  otherwise  finely  granular  and  occasionally 
vacuolated.  A  nucleus  and  endospores  were  not  demonstrated. 
The  capsule  did  not  give  the  cellulose  reaction.  From  the  round 
or  oval  bodies  segmented  mycelial  threads  developed,  on  which  in 
turn  new  spherical  organisms  or  conidia  formed.  This  observation 
estabhshed  the  unity  of  the  budding  cells  and  the  mycelial  threads. 
Through  several  generations  the  organism  varied  in  its  power  to 
form  mycelium,  at  times  growing  largely  by  budding  process. 

Buschke,  to  whom  Gilchrist  sent  a  culture,  found  that  upon 
potato  there  formed  an  aerial  mycelium,  which  developed  short 
lateral  branches,  without,  however,  forming  organs  of  fructifica- 
tion, as  in  hyphomycetes. 

Inoculation  of  the  tissue  into  mice  and  guinea-pigs  produced  no 
results,  serving  at  least  to  eliminate  tuberculosis.  Three  dogs,  a 
sheep,  and  a  horse  were  inoculated  intravenously  with  bouillon 
suspensions  of  the  organism.  It  was  common  for  them  to  develop 
nasal  discharges;  also  sinuses  at  the  point  of  inoculation.  They 
were  killed  in  from  one  to  two  months,  and  in  all  were  found  vari- 
ously sized  tubercle-like  nodules  in  the  lungs  and  mediastinal  l3^mph 
glands.  Cultures  of  the  blastomyces  were  obtained  from  the  lung 
lesions  except  in  one  dog  and  the  horse.  One  intra-abdominal 
inoculation  into  a  guinea-pig  proved  negative;  another  developed 
testicular  abscesses,  which  discharged  and  from  which  the  organism 
was  obtained  in  pure  culture.  Organisms  reclaimed  from  inocu- 
lated animals  were  inoculated  into  a  pig,  guinea-pigs,  and  mice, 
successes  and  failures  of  infection  being  about  equally  divided. 


40  Contributions  to  Medical  Science 

Attempts  to  produce  lesions  in  the  skin  of  animals,  resembling 
those  of  the  patient,  failed.  In  speaking  of  the  lung  lesions  in 
animals,  the  authors  said  that  "the  general  appearances  were  those 
of  a  chronic  inflammation,  and  one  would  not  be  hkely  to  regard 
the  lesions  as  those  of  a  neoplasm."  In  some  instances  they  were 
"typical  pseudo-tubercles  with  caseation."  MyceKum  was  not 
produced  in  any  of  the  lesions  in  animals  nor  in  the  disease  of  the 
patient. 

In  their  second  report,  in  speaking  of  the  classification  of  their 
organism,  they  say:  "In  our  preliminary  communications  we 
classed  our  organism  as  an  oidium,  for  the  following  reasons: 
the  organism  did  not  ferment  glucose,  saccharose,  or  lactose,  and, 
although  it  only  developed  by  gemmation  in  all  the  tissues,  human 
and  animal,  yet  upon  artificial  culture  media  it  developed  mycelia 
with  the  formation  of  conidia.  After  consulting  Brefield's  writings, 
and  also  after  a  personal  conference  with  Dr.  Irwin  Smith,  of 
Washington,  an  acknowledged  authority  in  mycology,  we  were 
inclined  to  consider  the  parasite  as  an  oidium.  On  further  investi- 
gation, we  find  that  mycologists  are  not  at  all  certain  about  the 
classification  of  either  the  yeast  fungi  or  the  oidia.  We  find  that 
there  are  a  number  of  wild  yeasts  which  do  not  ferment  sugar  and 
which  form  myceUum  on  the  usual  bacteriological  media.  In 
accordance  with  the  prevailing  custom  in  the  nomenclature  of 
organisms  presenting  characters  similar  to  ours,  we  now  prefer 
to  designate  the  parasite  as  a  blastomyces  rather  than  an  oidium." 
Accordingly  they  applied  the  name  "Blastomyces  derma titidis " 
to  the  organism. 

3.  This  case  was  described  by  H.  G.  Wells  in  March,  1898. 
The  patient  was  a  healthy  farmer,  forty  years  old,  and  a  native  of 
Iowa.  The  family  history  was  negative,  and  the  general  physical 
condition  of  the  patient  good.  The  disease  began  eleven  years 
pre\iously  as  a  pimple  on  the  dorsal  skin  of  the  left  little  finger. 
Tliis  ulcerated  and  extended  gradually  until  the  whole  dorsum  of  the 
hand  was  covered,  the  palm  being  avoided. 

The  whole  lesion  was  removed  and  the  defect  remedied  by  a 
plastic  procedure. 

Cultures  yielded  saprophytic  bacilli  and  pyogenic  cocci.    A  piece 


Oidiomycosis  of  the  Skin  and  Its  Fungi  41 

of  fresh  tissue  placed  subcutaneously  in  a  rabbit  caused  an  abscess 
the  size  of  a  hickory  nut,  which  discharged  spontaneously  on  the 
ninth  day.  From  the  resulting  sinus  white  pus  could  be  squeezed. 
A  second  rabbit  was  inoculated  with  this  pus.  An  indurated 
node  formed,  which  subsided  without  suppuration.  Repeated 
examinations  of  the  pus  failed  to  reveal  yeast  cells,  and  cultures 
yielded  Staphylococcus  pyogenes  aureus.  Microscopically,  the 
tissue  corresponded  closely  to  the  cases  of  Gilchrist,  and  Gilchrist 
and  Stokes.  Epithelial  hyperplasia,  intra-epithelial  abscesses, 
dense  infiltration  of  the  cutis,  giant  cell  formation,  and  the  presence 
of  a  capsulated  budding  fungus  were  characteristic.  The  abscesses 
contained  one  or  more  parasites.  Dr.  Wells  notes  that  in  some  of 
the  intra-epithelial  abscesses,  "having  no  communication  with 
corium,  tubercle-like  giant  cells  are  found,  which  must  have  been 
formed  either  from  the  epithelium  or  from  the  cells  that  have 
wandered  in."  Numerous  giant  cells  of  the  tuberculous  type, 
often  vacuolated,  were  found  in  the  corium.  However,  "none  of 
the  characteristic  zones  of  tubercular  foci  are  found  about  the 
giant  cells."  Tubercle  bacilli  could  not  be  demonstrated.  The 
number  of  single  and  budding  organisms  found  within  giant  cells 
was  remarkable.  The  structure  of  the  organism  was  given  as 
follows,  from  without  inward:  First,  a  capsule;  second,  a  trans- 
parent zone;  third,  a  central  protoplasm;  fourth,  a  vacuole 
within  the  protoplasm.  The  vacuole  was  present  inconstantly. 
Typical  budding  occurred. 

Plasma  cells  have  been  demonstrated  recently  in  large  numbers 
in  the  sections. 

It  is  to  be  noted  that  the  subcutaneous  implantation  of  the 
human  tissue  into  a  rabbit  produced  lesions  identical  with  those 
observed  in  animal  experiments  by  other  writers  using  pure  cul- 
tures of  "blastomycetes."  This  justifies  the  suspicion  that  a  fungus 
of  this  sort  caused  the  swellings  and  abscesses. 

4.  Reported  by  Hessler  in  August,  1898. 

In  October,  1897,  the  patient  was  cut  below  the  chin  while 
being  shaved  by  a  barber.  The  wound  healed  in  a  few  days,  but 
a  red  papule  soon  developed.  It  was  about  the  size  of  a  half-grain 
of  wheat,  hard,  elevated,  and  remained  stationary  for  almost  three 


42  Contributions  to  Medical  Science 

months.  It  increased  to  pea-size  and  suppurated.  No  pus  organ- 
isms were  found  in  smear  preparations,  but  there  were  large  budding 
cells,  which,  it  is  stated,  were  largely  within  leukocytes. 

Agar  inoculations  gave  pure  cultures  of  a  large  budding  cell 
"in  a  few  days."  On  the  surface  of  glycerin  agar  stabs  a  dirty 
white  mass  developed,  ''consisting  of  a  dense  network  of  fibers, 
with  cells  of  various  sizes."  The  organism  grew  poorly  in  plain 
glucose  solutions,  "but  abundantly  on  the  addition  of  a  little 
extract  of  beef  and  without  the  formation  of  carbonic  acid."  It 
was  stated  that  only  on  solid  media  did  fungus  threads  appear 
"after  days  or  weeks." 

The  organism  was  slightly  oval,  with  a  clear  outer  envelope, 
and  somewhat  smaller  than  the  yeast  described  by  Gilchrist. 
Welch  considered  it  a  blastomyces. 

5.  Reported  by  Hyde,  Hektoen,  and  Bevan  in  1898;  Hektoen 
in  1899  gave  an  additional  detailed  report  on  the  organism. 

The  patient  was  a  Hollander,  fifty-six  years  old;  his  general 
health  was  poor. 

The  disease  began  four  years  previously  as  a  reddish  spot  on 
the  right  leg,  nearly  the  whole  anterior  surface  being  finally  covered. 
The  case  was  regarded  as  one  of  tuberculosis  until  Hektoen  demon- 
strated budding  organisms  in  frozen  sections. 

The  histology  was  that  of  previous  cases;  epithehal  hyper- 
plasia, intra-epithelial  and  subcutaneous  miliary  abscesses,  dense 
infiltration  of  the  cutis,  and  the  presence  of  a  characteristic  organism 
being  the  principal  features.  There  were  isolated  epithehal 
whorls,  partially  cornified.  The  abscesses  were  not  different 
from  those  described  by  Gilchrist.  Plasma  cells  are  mentioned 
for  the  first  time  in  connection  with  this  disease.  Mulberry-Hke 
masses  of  hyalin  bodies  are  described,  one  occurring  in  a  cell  with 
a  plasma-cell-like  nucleus.  The  organism  measured  from  ten  to 
twelve  microns  in  diameter  and  presented  the  same  structural 
features  found  in  those  of  previous  cases.  It  was  not  found  in 
the  cutis,  nor  within  giant  or  epithelial  cells,  but  frequently  in  the 
intra-epitheUal  abscesses.  Budding  forms  were  numerous.  Methyl- 
ene blue  was  considered  the  best  stain  for  the  parasite,  the  capsule 
staining  deeply,  and  the  protoplasm  a  fighter  blue. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  43 

In  cultures  a  yeast  fungus  occurred  constantly,  but  intimately 
associated  with  a  pseudo-diphtheria  bacillus.  After  much  diffi- 
culty the  fungus  was  separated.  However,  further  confusion 
resulted  by  the  development  of  a  small  budding  organism,  which 
was  a  Httle  larger  than  a  large  coccus.  This  was  proved  satis- 
factorily to  be  a  form  of  the  larger  yeast  plant.  On  agar  and 
gelatin  plates,  distinct,  coarsely  granular  colonies  appeared  in 
twenty-four  hours.  Individual  cells  could  be  recognized  with  low 
magnification.  Threads  did  not  shoot  out,  as  happened  in  similar 
cultures  of  Gilchrist's  organism.  Multiplication  by  budding 
occurred  in  all  media.  On  translucent  solid  media,  abundant, 
feathery,  cone-shaped  down-growths,  resembling  those  seen  in 
some  varieties  of  ray  fungi,  appeared  in  from  eight  to  fourteen 
days.  This  also  occurred  in  stab  cultures.  Yellowish-brown 
pigment  formed  around  the  spherical  cells  on  plain  agar.  No  acid 
was  produced,  and  when  the  organism  was  first  described  no 
fermentation  occurred  in  lactose,  dextrose,  and  saccharose  solu- 
tions. (It  has  been  demonstrated  lately  that  the  ability  to  ferment 
maltose  and  dextrose  solutions  has  been  acquired.)  No  indol  was 
formed. 

It  was  perhaps  a  little  smaller  in  cultures  than  in  tissue.  The 
capsule,  clear  zone,  and  central  protoplasm  were  recognized  in  the 
cultures.  Gradually,  on  a  part  of  the  culture  tube,  the  organisms 
became  larger  and  more  vacuolated.  In  liquid  media  a  small 
form,  one  to  five  microns  in  diameter,  frequently  formed.  They 
were  observed  to  give  rise  to  a  large  type  of  the  organism.  Myce- 
lium formed  frequently  in  liquid  media.  Buds  would  arise  from 
any  point  of  the  myceHum,  and  were  sessile  or  pedunculated. 

A  guinea-pig  inoculated  with  tissue  died  a  month  later,  the 
pseudo-diphtheria  bacillus  being  recovered.  The  liver  was  cir- 
rhotic and  necrotic,  these  changes  being  ascribed  to  the  bacillus. 

Two  white  rats  received  subcutaneous  inoculations  of  a  bouillon 
suspension.  Abscesses  resulted  from  which  the  fungus  was  recov- 
ered in  pure  culture. 

Two  gray  mice  were  inoculated.  One  died  after  five  days. 
Pure  cultures  of  the  blastomyces  were  obtained  from  the  local 
abscess,  but  cultures  from  the  internal  organs  were  sterile.     Staphy- 


44 


Contributions  to  Medical  Science 


lococcus  albus  was  found  in  the  abscess  and  in  all  the  internal 
organs  of  the  second  animal.  Two  inoculations  in  the  anterior 
chambers  of  the  eyes  of  rabbits  resulted  in  h>popyon,  and  on 
their  death,  forms  resembling  the  blastomyces  were  found  in  the 
pus. 

Inoculations  into  guinea-pigs  gave  rather  negative  results. 

A  white  rat  died  from  an  intra-abdominal  inoculation.  There 
were  several  foci  in  the  limgs,  in  which  "large,  round,  doubly 
contoured  bodies"  were  found. 

A  rabbit  inoculated  intravenously  was  killed  after  a  month.  In 
the  lungs  were  found  mihary  foci  of  granulation  tissue,  but  no 
blastomycetes.  An  intraperitoneal  inoculation  of  a  dog  proved 
negative. 

A  rabbit  received  an  intravenous  inoculation  of  four  cubic 
centimeters  of  a  bouillon  suspension.  Death  occurred  the  next 
night.  Smears  and  cultures  from  all  organs  yielded  the  blastomy- 
ces. On  section,  the  lungs  were  congested,  and  blastomycetes 
were  found  in  necrotic  areas.  The  liver  and  spleen  were  normal. 
In  the  kidneys  the  glomerular  and  tubular  epithehum  were  degen- 
erated, and  in  the  former,  bodies  resembhng  blastomycetes  were 
found. 

A  later  intravenous  inoculation  into  a  dog  resulted  in  its  death 
in  about  a  month.  There  were  "minute  foci  of  granulation  tissue 
throughout  the  lungs,  and  softened  cellular  areas  with  yellowish 
contents  in  the  medullary  pyramids  of  the  kidneys.  The  blastomy- 
cetes were  recovered  in  pure  growth  from  the  lungs  and  kidneys." 

Hektoen  comments:  "Our  organism  grows  much  more  rapidly 
than  the  one  of  Gilchrist  and  Stokes.  The  formation  of  mycelium 
is  not  nearly  so  marked  as  in  their  cultures,  in  which  were  not  seen 
the  pecuhar  down  and  out  growths  and  lateral  branchings,  nor  the 
pigment  formation  (on  agar-agar)  characteristic  of  the  blasto- 
myces now  described.  Both  organisms  correspond,  however,  in 
their  action  on  gelatin,  which  is  not  liquefied,  in  the  non-production 
of  indol,  and  in  the  complete  absence  of  fermentation  of  various 
sugars.  Morphologically  they  are  also  quite  or  nearly  alike. 
Gilchrist  and  Stokes  make  no  mention  of  such  great  variability 
in  the  size  as  observed  in  our  organism." 


Oidiomycosis  of  the  Skin  and  Its  Fungi  45 

6.  Reported  in  November,  1899,  by  Owens,  Eisendrath,  and 
Ready. 

The  patient  was  a  widow,  thirty-eight  years  old.  Four  years 
previously  a  swelling  had  appeared  on  the  antero-internal  aspect 
of  the  lower  third  of  the  left  thigh. 

On  April  29,  1900,  the  whole  lesion  was  removed.  The  subcu- 
taneous tissue  was  not  involved.  The  wound  was  successfully 
closed  with  skin  grafts. 

"Under  the  low  power,  the  resemblance  of  the  section  to  an 
epithelioma  is  most  striking."  Epithelial  down-growths  were  pro- 
nounced, and  many  of  them  contained  miliary  abscesses.  The 
cutis  was  densely  infiltrated.  The  organism  was  found  in  the 
epithelial  prolongations,  either  between  cells  or  within  giant  cells; 
to  a  less  degree  in  miliary  abscesses,  and  most  of  all  in  the  cutis, 
either  surrounded  by  infiltration  cells  or  within  giant  cells.  The 
organisms  were  capsulated,  contained  a  clear  zone  and  a  central 
protoplasm,  the  latter  sometimes  showing  vacuoles,  and  sometimes 
possessing  ''small  bodies  like  spores."  Budding  was  observed. 
The  best  stain  was  methylene  blue. 

A  guinea-pig  was  inoculated  subcutaneously  with  a  piece  of 
fresh  tissue.  An  abscess  appeared  in  eight  days,  and  on  the  twelfth 
day  discharged  a  "creamy,  viscid,  foul-smelling  material."  Blas- 
tomycetes  were  not  demonstrated  in  smear  preparations  of  this 
pus.  Cultures,  however,  yielded  blastomycetes,  which  were  much 
contaminated  by  bacilli  and  cocci.  The  organism  was  obtained 
in  a  pure  state  by  growing  the  impure  cultures  in  potassium  iodide 
solutions.  Contaminating  organisms  were  thus  eliminated.  The 
blastomyces  suffered  a  reduction  in  size  in  this  process,  the  former 
size  being  regained  by  successive  inoculations  on  favorable  media. 

As  far  as  described,  this  organism  is  identical  with  that  of 
the  Hyde,  Hektoen,  and  Bevan  case.  However,  the  character- 
istics were  not  fully  worked  out.  It  is  unfortunate  that  the  pure 
culture  was  obtained  through  the  medium  of  an  animal  inoculation 
rather  than  directly  from  the  human  tissue.  The  chance  for 
error  was  thus  greatly  increased. 

7.  By  Ludvig  Hektoen  in  December,  1899. 

"Mrs.  W.,  aged  sixty-four,  married,  with  an  uneventful  family 


46  Contributions  to  Medical  Science 

and  personal  history,  was  admitted  into  Professor  Murphy's  clinic 
November  lo,  1897,  on  account  of  a  lesion  of  the  right  leg.  When 
she  appeared  in  the  clinic  there  was  a  circular,  elevated,  cauhflower- 
like  mass,  as  large  as  a  silver  dollar,  with  an  irregularly  ulcerated 
and  red  surface.  The  growth  was  regarded  as  probably  car- 
cinomatous, and  removed." 

The  tissue  was  preserved  as  a  museum  specimen  and  later  used 
for  histological  study.  It  was  described  as  an  ovoid,  raised  area, 
four  by  five  centimeters  in  its  principal  diameters.  The  verrucous 
character  of  the  surface  gave  it  a  cauliflower  appearance.  The 
color  was  yellowish  and  showed  superficial  erosions.  On  section, 
the  yellowish  tissue  of  the  lesion  was  sharply  defined  from  the 
underlying  fatty  tissue.  Histologically  the  elevation  was  made 
up  largely  of  hyperplastic  epithehum,  which  formed  irregular 
and  branching  bands,  masses,  and  nests,  presenting  a  "marked 
resemblance  to  flat-celled  carcinoma."  The  rete  exhibited  the 
usual  leukocytic  infiltration  and  intra-epithelial  abscesses.  Miliary 
abscesses  also  were  found  in  the  cutis.  Plasma  cells,  lymphocytes, 
eosinophiles,  giant  cells  of  the  tuberculous  type,  and  new-formed 
vessels  containing  lymphocytes,  plasma  cells,  and  polymorpho- 
nuclear leukocytes  were  present  in  the  cutis.  Smaller  giant  cells 
were  found  in  the  miliary  abscesses  in  the  corium  and  epithelium. 
The  organisms,  "typical,  doubly  contoured  blastomycetes,"  occur 
in  the  miHary  abscesses,  in  the  connective  tissue  of  the  corium,  and 
occasionally  in  giant  cells.  "The  organisms  are  round,  about 
twelve  microns  in  diameter;  distinct  budding  forms  are  present; 
some  of  the  bodies  contain  one  or  more  vacuoles  of  irregular  size 

and  outHne Some  organisms  are  stained  very  deeply  in  the 

methylene  blue  specimens." 

The  author  emphasizes  the  clinical  resemblance  of  the  growth 
to  carcinoma,  and  its  histological  resemblance  on  the  one  hand 
to  carcinoma  and  on  the  other  to  tuberculosis;  true  carcinoma 
may  some  day  be  found  in  connection  with  blastomycetic  dermatitis 
as  it  has  been  found  in  lupus.  He  considers  the  disease  well 
estabhshed,  and  points  to  the  probability  of  more  than  one  variety 
of  fungus  being  the  etiological  factor  in  different  cases. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  47 

8.  Anthony  and  Herzog  in  1900  reported  a  case  in  a  man  who 
gave  an  uneventful  personal  and  family  history. 

Of  the  histology  of  this  case,  it  is  sufficient  to  say  that  it  corre- 
sponds with  that  typical  of  "  blastomycetic "  dermatitis. 
Cultural  and  inoculation  experiments  are  not  recorded. 

9.  By  W.  E.  Coates,  February,  1900.  The  patient  was  a 
married  man,  thirty-eight  years  old,  giving  no  personal  or  family 
history  of  carcinoma,  tuberculosis,  or  syphilis.  His  general  health 
was  good. 

In  January,  1898,  the  knees  and  elbows  were  involved  in  acute, 
painful  rheumatism,  with  little  swelling.  Two  weeks  later  severe 
pains  began  in  the  chest,  and  he  was  confined  to  bed  for  three 
months  by  what  was  called  a  pleuro-pneumonia.  There  was  a 
cough  and  bloody  expectoration.  While  recovering  from  this 
illness,  a  small  nodule  appeared  on  the  lower  right  eyelid,  the  lesion 
reaching  the  size  of  a  bean  in  three  weeks,  when  it  was  removed. 
At  different  times  other  small  nodules  appeared  elsewhere. 

The  diagnosis  of  multiple  epithelioma  was  made  by  a  previous 
examiner,  based  upon  microscopic  examination  of  the  tumor  of 
the  eyelid.  Dr.  Coates,  however,  discovered  blastomycetes  in 
the  tumor  of  the  lip,  which  histologically  resembled  an  epithelioma. 
The  epithelium  had  proliferated  greatly  and  formed  large  masses 
and  pegs,  penetrating  the  cutis.  There  were  many  intra-epithelial 
abscesses  which  contained  giant  cells  and  a  budding,  capsulated 
cell  about  twelve  microns  in  diameter.  Smaller  spherical  bodies, 
taking  up  nuclear  stains,  the  author  thinks  are  small  forms  of  the 
organism,  although  the  difficulty  of  distinguishing  them  from  tissue 
detritus  is  admitted.  Some,  he  thinks,  are  small  spores.  The  large 
spherical  cells  are  considered  spore-bearing  bodies,  although  the 
evidence  for  this  belief  is  not  presented. 

There  are  also  miliary  abscesses  in  the  cutis,  containing  fungus 
cells  and  a  few  giant  cells.  The  cutis  is  densely  infiltrated  with 
leukocytes,  plasma-  and  mast-cells.  The  organism  does  not 
occur  in  giant  cells.  Tubercle  bacilli  could  not  be  demonstrated 
in  the  sections. 

The  author,  on  the  basis  of  a  chain  of  four  or  five  loosely  united 


48  Contributions  to  Medical  Science 

fungus  cells,  theorizes  quite  positively  as  to  hyphal  elements, 
mycelium,  sporing  cells,  and  secondary  spores.  He  considers  that 
the  intra-epithelial  abscess  is  "lined  by  deHcate  fungus  filaments." 
This  theory,  however,  is  not  satisfactorily  demonstrated. 

10.  At  the  meeting  of  the  Chicago  Academy  of  Medicine,  on 
January  12,  1900,  Dr.  Baldwin  presented  a  case  under  the  tentative 
diagnosis  of  epithelioma.  The  disease  occurred  in  a  Polish  woman, 
forty  years  old.  As  far  as  described,  the  beginning  of  a  lesion  was 
either  a  pimple  or  a  small  swelling,  "having  a  covering  like  tissue 
paper,"  the  rupture  of  which  resulted  in  a  discharge  of  pus.  The 
more  recent  lesions  measured  from  three  to  eight  centimeters  in 
diameter,  were  roughly  circular  or  oval,  elevated  from  two  to  five 
milHmeters,  and  had  a  fungoid  or  cauliflower-like  surface.  The 
borders  were  usually  more  elevated  than  the  centers  (which  were 
sometimes  decidedly  depressed),  and  surrounding  was  a  dark-red 
halo  of  elevated  skin.  There  was  a  little  oozing,  some  fetor, 
and  a  varying  amount  of  pain  and  tenderness.  The  older  lesions 
were  flatter  than  the  recent  ones.  There  was  no  glandular  enlarge- 
ment. Recently  some  anesthesia  had  developed  in  the  right  arm 
and  hand,  and  there  were  "rheumatic  pains  over  the  entire  left 
tibia."     No  history  of  syphilis  could  be  obtained. 

A  brief  histological  report  was  offered:  "Histologically,  an 
epithelioma;  a  typical  epithelial  cell  proliferation  into  the  deeper 
tissue.  Sections  were  stained  for  the  tubercle  bacilli,  but  none 
were  found,  though  stained  by  thionin,  methylene  blue,  and  the 
Klebs  and  triple  stain  for  protozoa;  some  deeply  staining  bodies, 
with  hyalin  rings  in  epithelial  masses,  were  probably  protozoa. 
A  few  organisms  were  found  with  double  contour." 

In  the  discussion  which  followed,  the  case  was  called  variously 
epithelioma,  lupus,  and  Mycosis  fungoides.  One  critic  observed 
that  "there  is  no  such  thing  as  blastomycetic  dermatitis  per  se, 
but  that  blastomycosis  is  found  secondarily  to  previous  lesions." 

It  is  unfortunate  that  attempts  were  not  made  to  cultivate  an 
organism  from  this  case,  and  that  a  more  detailed  histological 
report  was  not  offered. 

11.  Dr.  A.  W.  Brayton,  in  April,  1900,  reported  a  case  in  a 
livery-stable  employee,  fifty  years  old,  married. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  49 

The  description  of  the  lesions  is  brief:  "There  were  three  open 
and  undermining  ulcers  the  size  of  a  bean.  The  swollen  margins 
were  very  painful."  The  article  reports  that  Professor  Catherine 
Golden  found  "Blastomyces  dermatitidis "  in  the  sections,  and  that 
Dr.  W.  T.  S;  Dodds  cultivated  the  yeast  plant  on  wort-gelatin, 
subsequently  inoculating  a  guinea-pig,  which  two  weeks  later 
"showed  the  recognized  symptoms  of  blastomycetic  infection." 
The  yeast  plant  was  found  in  tissue  macerated  in  potassium 
hydrate  solutions.  The  communication  does  not  give  cultural 
or  histo-pathological  data. 

12.  Reported  to  the  International  Congress  of  Dermatology 
and  Syphilography  in  Paris,  August  2-9,  1900: 

The  patient  came  to  the  hospital  dispensary  of  Jefferson  Medical 
College  in  April,  1899.  He  had  been  scratched  or  bitten  by 
a  cat  on  the  back  of  the  right  hand.  This  wound  healed.  A 
few  weeks  later  a  pustule  appeared  at  the  point  of  traumatism. 
Gradual  extension  occurred,  eventually  covering  the  dorsum  of 
the  hand. 

Doctor  Rosenberger  gives  the  following  histological  and  cultural 
report :  The  pus  both  from  the  arm  and  the  hand  lesions  contained 
pus  cells,  bacilli,  cocci,  and  spherical  cells  possessing  buds.  In  sec- 
tions similar  spherical  budding  cells  were  found  deep  in  the  tissue, 
arranged  mostly  in  groups.  After  macerating  tissue  in  bouillon 
it  was  inoculated  on  glycerin  agar  and  a  liberal  culture  of  the 
large  spherical  budding  cell  obtained,  which  stained  homogene- 
ously. 

Other  cultural  and  histological  data  are  lacking.  It  was  con- 
sidered an  undoubted  case  of  blastomycosis  of  the  skin.  Complete 
cure  had  not  been  obtained. 

The  description  given  of  the  lesions  on  the  hand  answers  for 
that  of  typical  "blastomycetic"  dermatitis,  except  for  miliary 
abscesses.  No  mention  is  made  of  this  feature.  The  lesions  on 
the  arm,  however,  are  a  deviation  from  the  type  and  have  not  been 
observed  in  other  cases.  The  formation  of  an  abscess  which  is 
at  first  entirely  subcutaneous,  rupturing  later  at  several  points, 
causing  a  cribriform  appearance,  suggests  to  a  degree  the  sporothrix 
infections  of  Schenck  and  Hektoen. 


50  Contributions  to  Medical  Science 

13.  By  Isadore  Dyer,  January,  1901. 

The  patient  was  an  Englishwoman,  fifty-five  years  old. 

In  1898  a  growth  called  an  epithehoma  was  removed  from  the 
forehead  by  a  caustic  paste.  In  February,  1899,  "granulation 
sores"  appeared  on  the  backs  of  both  hands,  and  a  warty  growth 
on  the  face. 

The  author  emphasizes  the  clinical  resemblance  of  this  case  to 
"yaws." 

Gilchrist  studied  the  tissue  and  found  all  the  characteristic 
features  of  "blastomycetic"  dermatitis.  The  organisms  were  not 
numerous. 

Dr.  P.  E.  Archinard  after  many  experiments  obtained  an 
impure  culture  of  the  fungus,  which  was  sent  to  Gilchrist.  So 
far,  the  report  on  the  organism  is  incomplete.  Gilchrist  found 
spherical  and  oval  forms  and  a  branched  jointed  mycelium,  "which 
frequently  contained  spores."  It  is  remarked  in  a  general  way  that 
the  growth  on  media  is  not  at  all  like  that  of  the  Gilchrist-Stokes 
organism. 

The  photographs  and  description  of  the  case  are  typical  of 
"blastomycetic"  dermatitis,  and  speak  for  the  oidium-like  nature 
of  the  organism. 

14.  Presented  by  Frederick  G.  Harris  at  the  February,  1901, 
meeting  of  the  Chicago  Pathological  Society. 

The  patient  was  a  German  woman,  seventy-eight  years  old. 

Four  years  ago  a  pimple  formed  on  her  left  hip.  This  became 
roughened  and  denuded ;  it  extended  slowly,  and  was  accompanied 
by  moderate  itching. 

On  admission  to  the  Cook  County  Hospital  of  Chicago,  the 
clinical  diagnosis  of  syphilitic  ulcer  was  made. 

The  diagnosis  was  changed  to  epithehoma  after  a  small  piece 
had  been  removed  and  examined  microscopically.  Acting  on 
this,  the  area  was  excised  and  the  defect  covered  with  flaps. 

Microscopically  it  was  found  that  the  epitheHum  formed 
down-growths  some  distance  from  the  border,  these  increasing 
in  depth  and  complexity  as  the  border  was  approached.  The 
papillae  were  formed  by  extensions  of  the  corium,  which  were 
covered    with    the    proliferating    epithelium.     MiHary    abscesses 


Oidiomycosis  of  the  Skin  and  Its  Fungi  51 

occurred  in  the  hyperplastic  epidermis,  and  there  were  many 
epithelial  whorls. 

The  structure  and  contents  of  the  abscesses  are  similar  to 
those  in  other  cases.  Giant  cells  occurred  in  the  intra-epithehal 
abscesses  and  in  the  cutis.  The  cutis  was  densely  infiltrated  with 
polymorphonuclear  leukocytes  and  plasma  cells,  and  contained 
small  abscesses.  The  organism  was  found  in  intra-epithehal  and 
subcutaneous  abscesses  and  occasionally  in  the  granulation  tissue 
of  the  cutis  and  in  giant  cells.  It  formed  budding  pairs  and  groups 
of  three  or  four,  being  "often  surrounded  by  a  granular  material, 
which  took  the  eosin  stain."  They  were  not  found  free  in  the 
rete  nor  in  the  surface  discharge. 

A  small  tumor  which  had  existed  on  the  nose  for  twenty  years 
was  excised  and  foimd  to  be  a  benign  epithelioma,  of  the  tj^De 
which  originates  from  the  gland  ducts. 

Through  Dr.  Harris'  kindness  I  was  able  to  examine  his  speci- 
mens and  I  can  fully  corroborate  his  statements. 

4.    THE    PROTOZOIC    (?)     INFECTION    OF     WERNICKE     AND     OTHERS, 

ITS    RELATION    TO    SACCHAROMYCOSIS    HOMINIS    AND 

OIDIOMYCOSIS    OF    THE     SKIN. 

A  consideration  of  this  question  is  justified  here  because  of  the 
similarity  of  the  so-called  protozoic  infections  described  by  Posadas, 
Wernicke,  Rixford  and  Gilchrist,  and  others,  with  Busse's  Sac- 
charomycosis  hominis,  because  of  the  resemblance  of  the  skin 
lesions  in  these  two  diseases  to  oidiomycetic  dermatitis,  and 
because  some  of  the  organisms  concerned  are  virtually  identical. 

Wernicke  in  1892  reported  briefly  the  first  infection  of  this 
type,  accompanied  by  the  doubtful  diagnosis  of  Mycosis  fungoides. 
Rixford  and  Gilchrist,  however,  in  1896,  pubhshed  a  thorough 
description  of  two  cases,  estabhshing  a  rather  characteristic  histo- 
pathology,  and  concluding  that  the  organisms  were  true  sporozoa. 
They  gave  the  names  "  Coccidioides  immitis"  and  "  Coccidioides 
pyogenes"  to  the  organisms  in  the  two  cases,  because  of  their  close 
resemblance  to  coccidia  and  the  active  pyogenic  properties  of  one 
of  them.  Rixford  and  Thorne  in  1894,  and  Posadas,  Ophiils  and 
Moffitt,  and  D.  W.  Montgomery  in  1900  have  also  reported  cases. 


52  Contributions  to  Medical  Science 

Tropical  and  semi-tropical  countries  favor  the  infection,  judging 
from  the  histories  of  the  few  cases  reported.  Three  of  the  cases 
developed  the  disease  while  living  in  the  San  Joaquin  Valley, 
CaHfornia.  The  patients  have  been  men  in  early  or  middle  life. 
Heredity  seems  to  play  no  part,  and  the  occupations  have  been 
various.  It  seems  probable  that  the  infection  atrium  is  in  the 
lungs  in  most  cases,  although  in  Rixford  and  Gilchrist's  first 
case  it  is  fairly  positive  that  the  primary  seat  was  in  the  skin. 
The  disease  may  be  very  acute,  beginning  with  a  chill  and  fol- 
lowed by  rapid  lung  and  skin  involvement  (Ophiils  and  Mofiitt). 
A  cough  may  be  the  first  symptom  noticed,  serious  lung  and  skin 
involvement  soon  following  (D.  W.  Montgomery);  or  skin  lesions 
may  appear  before  visceral  involvement  is  observed  (Rixford  and 
Gilchrist's  first  case  and  Posadas'  case).  In  Rixford  and  Gil- 
christ's second  case  the  general  infection  was  fulminant,  following 
violent  hematemesis.  After  these  symptoms  of  general  invasion, 
emaciation  and  asthenia  follow  quite  rapidly;  the  temperature 
may  rise  to  101-103°  F.  in  the  evening  and  fall  to  almost  normal 
in  the  morning,  death  occurring  in  a  few  months.  Rixford  and 
Gilchrist's  first  case  lived  nine  or  ten  years  after  the  appearance  of 
the  first  skin  lesion,  and  about  a  year  after  the  first  hnrnphatic 
involvement.  Their  second  case  lived  four  months;  Ophiils  and 
Moffitt's  case,  only  three  months;  and  D.  W.  Montgomery's 
case,  about  a  year  after  the  appearance  of  the  first  symptoms, 
which  were  visceral  in  these  cases. 

The  skin  lesions  in  all  cases  have  certain  common  character- 
istics. They  may  begin  as  subcutaneous,  or  more  superficial 
abscesses;  or  granulomatous  growths  which  may  be  nodular 
(Wernicke,  Posadas,  D.  W.  Montgomery),  suggesting  Mycosis 
fungoides.  Eventually,  however,  in  all  cases  the  surfaces  are 
covered  with  thickly  set  large  papillae,  between  and  covering 
which  is  an  offensive  purulent  secretion.  The  surface  is  fungating 
or  cauliflower-like,  borders  usually  elevated  and  surrounded  by  a 
livid  and  moderately  indurated  narrow  zone,  and  the  centers  of 
larger  lesions  depressed  and  granulating.  Histologically  there  are 
great  epithelial  hyperplasia,  epidermal  and  subepidermal  abscesses, 
and  a  granulomatous  condition  of  the  corium  and  subcutaneous 


Oidiomycosis  of  the  Skin  and  Its  Fungi  53 

tissue,  marked  by  dense  infiltration,  giant  cells,  and  tuberculoid 
nodules.  The  organisms  are  present  in  vast  numbers  in  the 
surface  pus,  in  the  epidermal  and  subepidermal  abscesses,  and  in 
the  granulation  tissue  of  the  corium.  Lungs,  pleurae,  Kver,  kidneys, 
spleen,  bones,  suprarenals,  testicles,  and  the  lymph  glands  adjacent 
to  these  organs  have  been  found  invaded.  In  the  lungs  small  and 
large  abscesses,  miliary  nodules,  consohdation,  and  bronchopneu- 
monia occur.  The  lung  involvement  is  easily  recognizable  clini- 
cally. In  liver,  spleen,  kidneys,  testicles,  and  lymph  glands  there 
may  be  small  nodules,  or  abscesses  containing  cheesy  or  necrotic 
material.  In  Rixford  and  Gilchrist's  first  case  there  was  a  focus 
of  osteomyelitis  in  the  left  tibia  and  another  in  a  metacarpal  bone 
and  articulation,  the  joint  being  disorganized.  The  suppurating 
sinuses  led  to  sequestra.  Ophiils  and  Mofhtt  found  ostitis  and 
periostitis  of  the  frontal  bone  and  of  the  upper  part  of  the  left 
tibia,  with  suppuration  of  the  joint.  The  abscesses  and  nodules 
in  all  organs  consist  largely  of  the  protozoa  ( ?) ,  to  the  exclusion 
of  all  other  organisms. 

The  adult  parasite  in  tissues  is  unicellular,  varies  in  diameter  in 
different  cases  from  sixteen  to  thirty-five  microns,  possesses  a 
doubly  contoured  capsule  and  a  non-nucleated  protoplasm,  which 
gradually  is  transformed  into  a  hundred  or  more  structureless 
sporozoites  ( ?) .  These  escape  through  a  rupture  in  the  maternal 
capsule,  and  eventually  acquire  the  capsules  and  the  structure  of 
the  adult  cell.  No  budding  forms  have  been  mentioned.  One 
of  the  organisms  described  by  Rixford  and  Gilchrist  possessed 
vacuoles,  in  the  other  none  were  found.  This  is  the  life  history  in 
tissues  as  detailed  by  Rixford  and  Gilchrist,  and  by  Ophiils  and 
Mofhtt.  The  latter,  however,  cultivated  a  mould-fungus  from  all 
diseased  organs,  the  growths  being  unmixed  for  the  most  part. 
Pus  containing  organisms  was  mounted  in  a  hanging  drop  and 
the  shooting  out  of  mycelium  from  the  spherical  cells  noted.  Tissue 
and  pure  cultures  inoculated  into  animals  produced  conditions 
analogous  to  those  in  the  man,  the  spherical,  sporing  form  always 
appearing  in  tissues  and  the  mould-fungus  in  cultures.  These 
results  certainly  seem  to  establish  the  identity  of  the  forms  found 
in  the  tissues  and  the  culture. 


54  Contributions  to  Medical  Science 

There  seems  to  be  little  room  to  doubt  that  the  case  of  Ophiils 
and  Moffitt  is  identical  with  the  so-called  "protozoic"  disease 
of  Wernicke  and  others.  Then,  it  must  be  asked,  why  did  Rixford 
and  Gilchrist  fail  to  obtain  cultures  in  their  cases?  In  answer 
it  may  be  pointed  out  that  the  identical  state  of  affairs  is  seen 
in  "blastomycetic"  dermatitis.  The  organisms  in  two  cases  of 
"blastomycetic"  dermatitis  may  be  quite  similar,  yet  the  cultural 
pecuHarities  of  the  two  may  differ  widely,  one  growing  readily  on 
all  ordinary  media,  and  another  being  at  first  cultivated  only  on  a 
special  medium.  After  a  period  of  artificial  nourishment,  its 
adaptabiUty  may  make  it  possible  for  growth  to  occur  on  media 
which  at  first  were  unfavorable.  In  cases  where  an  organism 
could  not  be  cultivated,  the  nutrient  requirements  have  probably 
not  been  met. 

Although  it  is  anticipating  somewhat,  it  seems  proper  to  consider 
briefly  in  this  place  the  interrelationship  of  the  three  diseases 
which  have  been  summarized. 

Comparison  of  Busse's  case  with  the  so-called  protozoic  infec- 
tions shows  a  close  clinical  and  anatomical  analogy.  In  both  are 
found  a  chronic  pyemia,  eventually  ending  fatally,  characterized 
by  multiple  abscesses  and  miliary  or  larger  nodules  in  nearly  all 
viscera,  and  by  a  clinical  course  similar  to  general  miUary  tuber- 
culosis. In  both  the  lesions  contain  vast  numbers  of  parasites, 
and  the  purulent  secretions  Ukewise  are  heavily  laden  with  organ- 
isms. There  are  slight  differences  in  the  histology  of  the  skin 
lesions,  but  the  visceral  conditions  are  virtually  identical.  The 
bone  involvement  is  similar.  The  difference  noted  in  the  organisms 
concerned  is  not  sufficiently  great,  moreover,  to  warrant  a  wide 
separation  of  the  two  conditions,  as  illustrated  again  by  referring 
to  "blastomycetic"  dermatitis.  In  two  of  the  cases  of  the  latter 
disease,  identical  in  their  clinical  and  pathological  findings,  I  found 
two  different  organisms  in  the  cultures,  one  closely  related  to  that 
in  Busse's  case  (appearing  largely  as  a  budding  fungus),  and  the 
other  closely  related  to  that  in  Ophiils  and  Mofl&tt's  case  (growing 
as  a  mould-fungus).  In  other  words,  there  are  no  greater  differ- 
ences between  the  two  cases  of  fungus  septico-pyemia  reported 
by  Busse,  and  Ophiils  and  Moffitt,  respectively,   than  between 


Oidiomycosis  of  the  Skin  and  Its  Fungi  55 

Cases  I  and  VII  of  "  blastomycetic "  dermatitis,  in  respect  either 
to  clinical,  pathological,  or  cultural  findings. 

That  oidial  or  blastomycetic  dermatitis  is  related  to  the  so- 
called  protozoic  infections  and  to  Busse's  Saccharomycosis  hominis 
seems  extremely  probable,  particularly  when  the  conditions 
existing  in  Case  V  of  this  series  are  studied.  It  seems  probable 
that  the  first-named  disease  seldom  becomes  a  generahzed  in- 
fection. 

The  difference  between  the  skin  lesions  of  the  "protozoic" 
disease  and  of  ''blastomycetic"  dermatitis  may  be  considered 
as  one  of  virulence  purely.  In  the  former  more  pus  is  formed, 
and  the  organisms  are  present  in  much  larger  numbers.  There 
are  the  same  papilliform  lesions,  the  same  minute  changes  in 
epidermis  and  corium,  and  the  same  relation  of  the  organisms  to 
the  lesions.  The  essential  difference  in  the  organisms  is  that 
those  of  the  so-called  "protozoic"  infections  have  not  been  observed 
to  proliferate  by  budding,  while  in  blastomycetic  dermatitis  this 
is  the  only  proved  method  of  proliferation  in  tissues.  The  former 
appear  to  produce  endogenous  spores;  several  organisms  of  the 
latter  disease  (Cases  III,  VI,  VII,  VIII,  IX,  and  X)  produce 
similar  endocellular  bodies.  The  organism  described  by  Ophiils 
and  Moffitt  is  a  mould-fungus;  five  of  ours  are  mould-fungi. 
Apparently,  then,  the  differences  may  be  those  of  adaptation,  or, 
at  the  most,  specific  variations. 

Between  blastomycetic  dermatitis  and  Busse's  Saccharomycosis 
hominis  there  are  certain  differences.  The  former  so  far  is  a 
local  disease;  Busse's  was  a  general  infection.  In  the  skin 
lesions  of  the  latter  the  papillomatous  surface  did  not  develop 
and  necrosis  was  extensive.  According  to  Buschke  the  parasite 
destroyed  the  epithelium  rather  than  caused  hyperplasia.  We 
infer  that  intra-epitheHal  abscesses  did  not  form,  as  the  authors 
do  not  speak  of  them.  If  that  is  true,  it  constitutes  an 
essential  histological  difference  between  the  two  processes  in  the 
skin. 

As  shown  in  the  table  (p.  148),  Busse's  organisms  differ  only 
in  a  few  details  from  a  whole  group  isolated  from  cases  of 
blastomycetic  dermatitis. 


56  Contributions  to  Medical  Science 

SUMMARY. 

The  disease  called  a  protozoic  infection  by  Wernicke,  Rixford 
and  Gilchrist,  and  others  seems  identical  with  the  case  reported 
by  Ophiils  and  Moffitt,  from  which  the  latter  cultivated  a  mould- 
fungus.  The  disease  described  by  Busse  as  Saccharomycosis 
hominis  is  identical  clinically  and  anatomically  with  the  Ophiils 
and  Mofl&tt  case,  the  difference  in  the  skin  lesions  not  being  essential, 
and  the  two  organisms  being  separated  by  specific  rather  than 
generic  differences.  Oidial  dermatitis,  called  by  Gilchrist  *'blas- 
tomycetic  dermatitis,"  differs  from  both  the  above  in  being  purely 
a  local  disease,  and  in  having  a  relatively  small  number  of  organisms 
in  the  purulent  secretions  and  skin  lesions.  It  resembles  the  skin 
lesions  of  the  so-called  protozoic  infection  both  grossly  and  histologi- 
cally. It  differs  from  the  skin  lesions  of  Saccharomycosis  hominis 
in  that  the  latter  exhibits  an  excess  of  destruction  over  hyperplasia; 
and  its  various  organisms  differ  from  those  of  the  protozoic  disease 
and  Saccharomycosis  hominis  in  no  greater  degree  than  they 
differ  among  themselves;  hence  the  differences  between  these 
diseases  may  all  be  explainable  on  the  score  of  specific  variations 
of  the  fungi  concerned. 

III.    NEW  CASES  OF  OIDIOMYCOSIS  OF  THE  SKIN. 

Case  I.^ — Clinical  history  (Dr.  Montgomery). — J.  H.  C.  pre- 
sented himself  August  i8,  1899,  with  a  distinct  tumor-Uke  swelling 
of  the  lower  lip.     He  was  forty-five  years  old. 

Examination  showed  an  irregularly  rounded  tumor  of  the  lip, 
situated  a  little  to  the  left  of  the  median  line,  averaging  about  two 
centimeters  in  diameter. 

On  his  second  \isit  the  man  stated  that  the  corn  crop  of  the 
previous  year,  in  his  locality,  had  been  unusually  subject  to  a 
form  of  "dry  rot,"  producing  a  fine,  brown,  very  light  powder 
between  the  husk  or  kernel  and  the  cob.  He  had  handled  some 
of  this  grain,  and  said  that  many  cattle  and  some  horses  had  died 
after  eating  corn  so  diseased  that  had  been  left  in  the  fields.  He 
further  stated  that  he  was  in  the  habit  of  chewing  and  biting  grain. 

»  A  brief  report  of  Cases  I  and  II  has  been  made  (Montgomery  and  Ricketts),  Jour.  Cutaneous 
and  Genito-Urinary  Diseases,  igoi. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  57 

The  improvement  under  iodide  of  potassium  on  the  whole  was 
so  slight  that  an  operation  was  decided  upon,  and  on  November 
4  the  tumor  was  removed. 

Cultures. — Deeply  situated  fragments  of  tissue  were  excised 
aseptically,  teased,  and  inoculated  on  ordinary  culture  media. 
At  the  end  of  twenty-four  hours  small,  elevated  colonies,  with 
a  moist,  whitish,  and  gHstening  surface,  were  observed  on  agar  and 
blood-serum  slants;  and  on  potato  small,  dry,  white  elevations, 
which  in  a  few  days  grew  directly  into  the  air  in  the  shape  of  fine 
granular  shafts.  Two  other  varieties  of  colonies  appeared,  which 
proved  to  be  staphylococci  and  streptococci. 

Smears  from  the  colonies  first  mentioned,  stained  with  LoefHer's 
methylene  blue,  disclosed  a  large  micro-organism  of  varied  mor- 
phology. The  predominating  form  was  oval,  and  to  many  of  these 
were  attached  smaller,  apparently  budding  daughter  cells.  Short 
segmented  threads  were  common.  Both  the  oval  forms  and  threads 
had  a  delicate,  translucent  capsule  and  a  central  substance,  which 
stained  irregularly.  The  single  forms  averaged  seven-eighths 
micron  in  diameter,  though  there  was  great  variation  in  size.  The 
colonies  of  the  fungus  were  greatly  in  excess  of  those  of  all  other 
organisms  combined.  Its  biological  characters  were  studied  on 
various  culture  media. 

Ox-blood  serum. — A  broad,  grayish-white  line  develops  within 
twenty-four  hours,  which  grows  slowly  at  the  room  temperature, 
and  more  rapidly  when  in  the  brood-oven.  Within  five  days  the 
growth  is  elevated,  substantial,  moist,  one  and  one-half  millimeters 
broad,  and  has  a  smooth,  glistening,  white  surface,  marked  by 
scattered  pin-point  elevations  and  depressions.  The  edge  is 
irregular  and  sends  out  hnear  projections,  straight  or  curved, 
which  may  possess  small,  knob-like  extremities.  The  growth, 
under  the  microscope,  presents  spherical  and  oval  adults,  budding 
forms,  and  occasional  elongated  cells  (abortive  myceUum).  There 
are  some  particularly  large  cells  (involution  forms),  of  irregular 
contour,  much  vacuolated,  and  with  very  little  chromophilic 
substance. 

Agar  slants. — On  the  agars  (glycerin,  glucose,  maltose,  plain, 
and  beer-wort  agar)  the  growth  is  more  rapid  than  on  blood-senmi. 


58  Contributions  to  Medical  Science 

It  grows  least  readily  on  plain  agar.  There  are  the  same  surface 
appearances  as  on  blood-serum,  but  the  edge  is  flat  and  smooth. 
After  three  or  four  weeks  the  surface  becomes  more  uneven,  appear- 
ing as  if  bubbles  of  gas  had  burst,  making  circular  rents.  This 
is  most  noticeable  on  glucose  and  beer-wort  agar.  Fine  rays 
penetrate  the  medium  vertically.  Microscopically  the  agar 
growths  dififer  from  the  blood-serum  only  in  forming  more  myce- 
lium. 

Specimens  from  a  maltose  agar  tube  show  polychromatism, 
certain  cells  exhibiting  red  granules  with  alkaline  methylene  blue. 
Young  cells  stain  deeply  and  uniformly;  older  cells  have  one  or 
more  deeply  staining  granules;  the  oldest  and  largest  may  either 
stain  deeply  or  not  at  all. 

On  -f  lo  oxalic  acid  maltose  agar,  development  is  slow,  and  the 
surface  has  a  moist,  glassy,  yellowish- white  appearance.  No 
mycelium,  not  even  elongated  cylindrical  cells.  After  one  hour 
in  a  hanging  drop  of  tap  water  mycehal  segments  began  to  shoot 
out  and  budding  was  stimulated.  The  central  protoplasm  is 
sometimes  differentiated  into  a  spherical,  slightly  tinted,  nucleus- 
like substance,  which  may  or  may  not  contain  one  or  two  fat-hke 
globules. 

On  a  glycerin  agar  slant,  four  months  and  ten  days  old,  the 
surface  is  Kght  brown  and  shows  circular  rents.  Mounted  in  a 
KOH  solution,  the  spherical  cells  are  from  2  to  i6  microns  in  diam- 
eter. The  protoplasm  is  granular,  and  contains  many  fat-Hke 
globules.  In  the  largest  cells  the  capsule  is  from  1.5  to  2  microns 
thick,  but  the  accidental  capsule  seen  by  Busse  and  Curtis  in  their 
organisms  does  not  occur.  There  are  mycehal  fragments  of  similar 
structure.  The  reverse  surface  of  agar  slants  does  not  show  a 
golden-brown  color. 

Glucose  agar  stab. — No  growth  occurs  in  the  anaerobic  stab. 
In  the  aerobic,  the  line  is  gradually  marked  by  a  whitish  growth, 
which  soon  sends  deHcate  horizontal  rays  into  the  medium.  This 
is  seen  more  clearly  in  the  gelatin  stab. 

Glucose  agar  plate. — Within  twenty-four  hours  small  white 
colonies  develop,  which  have  characteristics  identical  with  those 
seen  on  the  original  agar  slants.     Under  the  low  power  they  appear 


Oidiomycosis  of  the  Skin  and  Its  Fungi  59 

granular,  and  around  the  border  minute  globular  forms  are  seen, 
closely  packed,  but  sharply  defined.  No  mycelium.  The  buried 
colonies  are  smaller  than  those  on  the  surface. 

Bouillon  gelatin  stab. — ^A  white,  finely  granular  growth  occurs 
along  the  line  within  twenty-four  hours.  Gradually  deHcate 
threads  penetrate  the  medium  transversely,  and  eventually  become 
densely  packed.  The  medium  is  not  liquefied.  A  plum-decoction 
gelatin,  however,  was  liquefied  slowly. 

Potato. — At  the  end  of  twenty-four  hours  the  growth  is  con- 
spicuous, and  proceeds  rapidly;  is  coarsely  granular,  elevated,  and 
tends  to  heap  up  rather  than  extend  over  the  surface  of  the  medium. 
When  the  potato  is  fresh,  the  growth  has  a  moist,  grayish-white 
color,  but  later  a  dry,  white,  powdery  appearance  is  acquired. 
Many  large  vacuolated  and  granular  degeneration  forms  occur. 

Bouillon. — Growth  is  somewhat  more  rapid  in  two  per  cent 
glucose  than  in  plain  bouillon.  In  two  to  five  days,  at  brood- 
oven  temperature,  a  grayish- white,  fluffy  cloud  lies  at  the  bottom 
of  the  tube,  the  overlying  fluid  remaining  clear.  The  sediment  is 
easily  disseminated  by  shaking.  Microscopic  examination  shows 
mycelium  and  spherical  forms  promiscuously  mixed.  These  are 
best  studied  in  the  hanging  drop.  The  findings  are  similar  in 
maltose  bouillon. 

Hanging-drop  culture. — Growth  occurs  with  or  without  myce- 
lium. In  the  latter  event,  it  consists  of  ascus-like  cells,  which 
multiply  by  budding.  The  mycelium  is  segmented  and  branching. 
The  oldest  segments  are  from  four  to  six  microns  in  diameter, 
taper  toward  the  growing  end  of  the  thread,  and  are  more  or  less 
irregular  in  outline,  offering  a  multitude  of  small  projections,  from 
which  buds  or  conidia  have  recently  separated.  The  end  of  a 
thread  sometimes,  but  not  habitually,  develops  a  small  cluster  of 
conidia  (Fig.  13,  Plate  7).  The  segments  are  from  two  to  five 
times  as  long  as  broad.  The  capsule  is  more  delicate  than  that  of 
the  adult  round  organism.  Within  the  capsule  lies  a  relatively 
narrow  clear  zone,  and  centrally  the  finely  granular  protoplasm, 
which  may  be  vacuolated.  Whole  threads  are  sometimes  made 
up  of  short,  incomplete  segments.  Portions  of  threads  may  be 
made  up  of  globules  of  granular  protoplasm  in  chains,  surrounded 


6o  Contributions  to  Medical  Science 

and  separated  from  each  other  by  a  clear  substance,  the  whole 
chain  being  enveloped  in  a  cyUndrical  membrane.  Only  septa 
are  necessary  to  the  production  of  chains  of  spherical  forms,  and 
this  is  accompUshed  gradually.  The  round  or  oval  adult  organism 
(the  ascus-like  body)  sometimes  forms  chains,  giving  off  here  a 
bud  and  there  a  link  of  myceUum.  It  has  a  capsule,  a  clear  space 
internal  to  this,  and  a  granular  central  protoplasm,  which  is  some- 
times vacuolated.  The  budding  process  occurs  as  described  by 
Gilchrist  and  Hektoen.  The  daughter  cells  have  a  thin  membrane 
and  do  not  possess  a  clear  space  or  vacuoles.  Occasionally  a 
large  form  is  seen  with  several  daughter  cells  attached.  When 
separated,  they  each  leave  a  small  projection  at  the  point  of  attach- 
ment to  the  mother  cell.  Daughter  cells  may  multiply  before 
separating,  and  with  further  multipHcation  a  cluster  of  from  four 
to  twelve  organisms  may  form. 

On  old  agar  tubes  the  cells  are  eight  to  ten  microns  in  diameter, 
sUghtly  shrivelled,  and  much  vacuolated  (involution  forms).  The 
new  cells,  however,  growing  in  a  hanging  drop,  are  apiculate  and 
do  not  form  mycelium.  After  a  few  months'  Hfe  on  artificial 
media,  myceUum  almost  entirely  ceased  to  form,  prohferation 
taking  place  by  budding  of  the  spherical  or  oval  cells.  After 
inoculating  into  two  mice  successively  and  reclaiijiing  the  organism 
after  death  of  the  hosts,  myceHum  again  grew  freely.  A  minute, 
coccus-like  form  is  sometimes  seen  singly  or  in  groups  of  from  two 
to  eight,  and  exhibits  Brownian  movement  when  free.  They  are 
frequently  adherent  to  the  larger  forms.  Clusters  of  small  forms 
are  bound  together  by  a  gha-Uke  substance.  (In  succeeding 
cultures  this  type  failed  to  develop.)  Neither  endospores  nor 
nuclei  have  been  demonstrated. 

Litmus  milk. — A  Uberal  bottom  and  a  surface  growth  occur. 
The  reaction  is  not  changed  and  the  milk  is  not  coagulated. 

Fermentation  tests. — Glucose  and  maltose  solutions  ferment 
readily,  with  the  production  of  alcohol  and  COj;  lactose  and 
saccharose  solutions  are  not  fermented. 

Potassium  iodide  cultures. — The  organism  grows  liberally  in 
a  one  or  a  five  per  cent  solution.  An  amorphous  brown  pigment 
is  formed  in  the  bottom  of  the  tube.     Budding  is  quite  general, 


Oidiomycosis  of  the  Skin  and  Its  Fungi  6i 

but  there  is  no  mycelium,  A  small  amount  of  proliferation  took 
place  in  a  hanging  drop  of  a  50  per  cent  solution.  At  the  end  of 
two  weeks,  however,  these  organisms  had  died,  as  shown  by  a 
refusal  to  grow  on  favorable  media. 

Indol  test. — In  sugar-free  bouillon  prepared  by  the  Theobald 
Smith  method,  a  faint  indol  reaction  was  first  obtained  after  ten 
days.  During  the  next  ten  days  it  more  than  doubled  in  intensity. 
The  ordinary  forms  of  the  organism  grew  in  this  solution. 

Thermal  death  point. — 55°  C.  for  four  minutes  kills  the  organism. 
55°  C.  for  two  minutes  retards  growth  twenty-four  hours. 

HiSTOPATHOLOGY. — Alcohol,  Zenker's  fluid,  and  saturated  solu- 
tion of  bichloride  of  mercury  were  used  as  fixatives;  celloidin  and 
parafl&n  imbedding;  hematoxyhn  and  eosin,  eosin  and  methylene 
blue,  polychrome  methylene  blue,  the  Gram-Weigert  method, 
the  tubercle  bacillus  method,  acid  fuchsin,  and  other  stains  were 
used. 

Under  the  low  power  the  surface  contour  is  uneven,  with  occa- 
sional cross-sections  of  horizontally  placed  villiform  processes. 
A  varying  amount  of  debris  is  found  on  the  surface.  The  horny 
layer  is  either  entirely  lost,  or  composed  largely  of  flattened  nucle- 
ated cells,  and  not  sharply  defined  from  the  underlying  rete.  Infil- 
trating leukocytes  are  so  numerous  as  to  make  the  layer  almost 
indistinguishable  under  the  low  power.  Disintegrating  red  blood 
cells  He  in  small  masses  and  rows  between  the  layers  of  epithelial 
cells.  The  nuclei  of  the  infiltrating  cells  are  much  distorted  in 
accommodating  themselves  to  the  spaces  they  occupy.  At  times 
a  filamentous  or  branching  form  is  assumed,  which  gives  a  close 
resemblance  to  fungous  hyphae. 

The  prickle-cell  layer  has  no  constant  thickness  or  configu- 
ration. In  places  it  is  exposed,  and  often  only  one  or  two  layers 
of  rete  cells  protect  the  cutis.  However,  throughout  the  diseased 
area  there  is  an  enormous  proliferation,  which  has  led  to  the  forma- 
tion of  grotesque  epitheUal  processes,  which  extend  into  the  cutis 
to  varying  depths,  some  being  delicate,  others  constituting  large 
masses.  Counter-formations  of  papillary  tissue  are  closely  mingled 
with  the  epithelial  processes,  and  deformed  papillae  are  often  cut 
transversely,  appearing  as  islands  of  cutis  tissue.     None  of  the 


62  Contributions  to  Medical  Science 

epithelium  is  in  isolated  masses.  The  cells  constituting  the 
down-growths  are  often  stretched,  elongated,  spindle-form. 
Throughout,  the  individual  prickle-cells  are  large,  commonly 
vesiculated  (edematous),  and  the  prickles  conspicuous.  The 
nuclei  likewise  appear  swollen  and  edematous.  Mitoses  are  fre- 
quent in  the  deeper  portions.  The  granoplasm  in  certain  areas 
is  unevenly  distributed,  being  massed  in  the  deeper  portions  of 
the  cell.  It  stains  better  with  basic  than  with  acid  dyes.  Such 
an  area  gives  a  peculiar  terraced  appearance  to  the  field,  the 
"cobblestone"  appearance. 

EpitheHal  cells  with  two,  three,  or  even  four  vesicular  nuclei 
are  found,  not  always  at  points  of  marked  infiltration.  Mitoses 
are  seen  occasionally  in  such  cells.  Inclusion  of  one  epithelial  cell 
by  another  as  cited  by  Gilchrist  is  observed  frequently.  Occasion- 
ally an  epithehal  cell  has  included,  or  has  been  invaded  by,  a 
polymorphonuclear  leukocyte;  the  epithehal  nucleus  may  retract 
or  shrink  to  make  room  for  the  leukocyte  in  the  perinuclear  space. 

Polymorphonuclear  and  mononuclear  leukocytes,  eosinophiles, 
plasma-  and  mast-cells  are  found  in  the  rete,  the  last  three  occur- 
ring rarely.  The  polynuclears  and  mononuclears  are  more  numer- 
ous. In  certain  places  the  polynuclears  occur  in  distinct  groups, 
almost  to  the  exclusion  of  all  other  wandering  cells,  constituting 
intra-epithelial  abscesses.  The  distinguishing  feature  of  the 
abscesses  in  this  case  is  their  minute  size.  The  origin  may  be 
within  an  epithelial  cell,  which  eventually  is  destroyed  as  the 
leukocytes  multiply.  Included  thus  no  compression  effect  is  made 
on  surrounding  epithelial  cells  until  the  invaded  cell  is  destroyed. 
Originating  in  intra-epitheHal  spaces,  adjacent  prickle-cells  are 
flattened  to  varying  degrees,  and  the  prickles  are  lost  while  the 
abscess  is  still  minute.  The  flattening  process  continues  as  the 
abscess  increases  in  size,  so  that  eventually  the  surrounding  cells 
appear  as  fibers  in  which  nuclei  may  or  may  not  be  found.  Thus 
a  Hmiting  capsule  is  formed  of  several  concentrically  disposed 
layers  of  flattened  epithelial  cells.  The  more  remote  cells  are  less 
and  less  flattened.  Between  the  fiber-like  cells  nearest  the  abscess 
leukocytes  accumulate.  As  they  increase  in  numbers  the  flattened 
and  elongated  rete  cells  are  loosened  and  become  a  part  of  the 


Oidiomycosis  of  the  Skin  and  Its  Fungi  63 

abscess  contents.  As  such  they  are  relatively  long  threads,  with 
tapering  extremities,  having  elongated  flattened  nuclei,  and  some- 
times presenting  the  appearance  of  branching  because  of  close 
union  with  other  cells.  They  are  sometimes  not  unlike  translucent 
fungous  filaments.  Instead  of  flat  cells  the  wall  is  at  times  made 
up  of  cuboidal,  normally  shaped  rete  cells  which  may  become 
detached  and  form  part  of  the  abscess  contents.  Such  cells  become 
spherical,  their  protoplasm  granular,  and  strongly  acidophilic, 
their  nuclei  swollen  and  possessing  faint  staining  properties. 
Either  before  they  become  separated  from  the  wall  or  later  they 
may  enclose  leukocytes  and  other  epitheUal  cells.  When  a  poly- 
morphonuclear leukocyte  is  first  enveloped  its  protoplasm  is 
prominent  as  a  clear  hyaHn  mass,  imbedded  in  the  acidophilic 
protoplasm  of  the  epithelial  cell.  The  leukocytic  nucleus  stains 
deeply.  MultipUcation  takes  place  or  new  cells  are  added  until 
the  epithehal  cell  forms  a  mere  shell  around  the  included  cells.  The 
nucleus  is,  of  course,  much  flattened  out  in  the  surrounding  shell, 
but  still  surrounded  by  body  protoplasm.  One  epithelial  cell  may 
include  another  within  the  abscess,  and  either  the  including  or 
the  included  cell,  or  both,  may  have  enclosed  leukocytes  or  nuclear 
detritus.  Epithehal  cells  sometimes  enclose  a  large  number  of 
small  spherules  of  uniform  size  which  stain  indistinctly.  Their 
identity  is  uncertain. 

As  mentioned,  the  dense  infiltration  of  the  cutis  and  rete  in 
many  places  makes  it  difficult  to  separate  the  layers.  This  infiltra- 
tion extends  into  the  fatty  and  muscular  tissue.  There  is  no 
distinct  abscess  formation  in  the  cutis,  no  collection  of  polymor- 
phonuclear leukocytes  to  the  exclusion  of  other  cells,  notably 
plasma  cells;  and  in  no  place  are  the  minute  abscesses  of  the 
epidermis  seen  communicating  with  the  cutis.  Plasma  cells,  lym- 
phocytes, polymorphonuclear  leukocytes,  mast-cells,  and  eosino- 
philes  constitute  the  mass  of  infiltration.  In  addition  are  newly 
formed  blood  and  lymph  channels,  with  marked  congestion  of  the 
former.  New  fibrous  tissue  is  forming  at  the  periphery  of  the  lesion. 
Elastic  tissue  has  disappeared  from  the  areas  of  most  severe  inflam- 
mation, but  is  still  present  rather  freely  in  the  peripheral  portions. 
In  certain  places  the  polymorphonuclears  predominate;    healthy 


64  Contributions  to  Medical  Science 

connective-tissue  fibers  and  cells,  plasma  cells,  and  granular  ma- 
terial are  intermingled.  Mononuclears  are  scattered  more  diffusely 
than  the  polymorphonuclears  and  are  not  collected  into  decided 
groups.  They  are  commonly  more  numerous  in  the  midst  of 
plasma  cells.     Eosinophiles  are  insignificant  in  number. 

The  striking  feature  of  the  subcutaneous  tissue  is  the  number  of 
mast-  and  plasma-cells.  The  latter  occur  in  groups  and  columns 
and  usually  in  close  proximity  to  blood-vessels.  This  relationship 
is  not  constant,  however.  They  occupy  broad  areas  at  times, 
associated  with  other  cells,  particularly  mononuclear  leukocytes, 
mast-  and  connective- tissue  ceils.  The  plasma  cells  remain  at 
points  relatively  remote  from  the  foci  of  most  intense  inflammation. 
They  are  present  everywhere  in  the  periphery  of  the  lesion,  extend- 
ing at  times  in  small  groups  into  the  muscular  and  fatty  framework. 
Occasionally  they  are  seen  within  blood-vessels  where  the  plasma 
cell  morphology  is  not  always  complete  in  all  details.  For  example, 
it  is  not  uncommon  to  find  in  blood-vessels  cells  with  the  typical 
plasma  cell  nucleus,  but  with  a  protoplasm  which  stains  very  faintly, 
or  not  at  all,  with  alkaline  methylene  blue.  One  typical  plasma  cell 
was  seen  emerging  through  the  vessel  wall. 

In  intimate  association  with  plasma  cells  everywhere  is  newly 
forming  fibrous  tissue.  Another  conspicuous  property  of  the 
plasma  cell  is  the  so-called  hyalin  degeneration. 

Mast-cells  are  thickly  and  widely  distributed.  Like  the  plasma 
cell,  they  are  absent  from  intensely  inflamed  areas.  A  certain 
tj^e  (leukocytic?)  is  found  in  close  proximity  to  blood-vessels. 
Another  variety  (the  connective-tissue  cell  type)  occurs  in  the 
periglandular  and  perifollicular  tissue,  but  is  found  also  in  the 
stroma  of  adipose  and  muscle  tissue.  Furthermore,  a  form  inter- 
mediate between  plasma-  and  mast-cells  seems  probable.  This 
cell  has  a  distribution  similar  to  the  type  just  mentioned.  That 
the  mast-cell  may  be  endowed  with  motile  properties  is  indicated 
by  its  occasional  presence  in  the  rete. 

Giant  cells  of  the  tubercular  type  are  seen  commonly  in  groups 
in  the  areas  of  most  dense  infiltration.  They  often  contain  from 
one  to  six  or  eight  vacuoles,  usually  oval  in  form.  It  has  not  been 
possible  to  demonstrate  positively  that  these  spaces  were  formerly 


Oidiomycosis  of  the  Skin  and  Its  Fungi  65 

occupied  by  organisms.  From  their  almost  constant  oval  form, 
however,  this  seems  probable. 

Hundreds  of  sections  have  been  searched  in  the  vain  hope  of 
finding  in  the  hardened  tissue  the  organism  which  was  cultivated 
from  the  fresh  so  readily.  Single  and  combined  stains,  with 
diverse  methods  of  fixation  and  differentiation,  have  been  used. 
One  would  expect  to  find  the  causal  agent,  of  course,  where  the 
fray  is  fiercest.  The  miliary  abscesses  may  be  taken  as  the  cul- 
minative  effort  of  resistance  on  the  part  of  the  tissues  against  the 
invading  organism.  Many  extra-  and  intra-cellular  forms  have 
been  found  which  might  represent  the  organism.  The  most 
common  is  a  spherical  body,  from  one  to  three  microns  in  diameter, 
which  occurs  in  groups  in  phagocytic  cells  or  free  within  abscesses. 
They  stain  with  hematoxyhn,  by  Gram's  method,  with  acid  fuchsin, 
and  a  blue  violet  with  polychrome  methylene  blue.  When  it  is 
remembered  that  a  small  form  of  the  organism  was  developed  in 
cultures,  and  furthermore  that  the  morphology  of  these  organisms 
is  variable,  the  temptation  is  strong  to  rest  with  the  finding  of  such 
a  form  as  mentioned  above,  particularly  since  it  occurs  where  one 
expects  to  find  the  organism.  However,  the  morphological  evidence 
is  not  sufficient,  as  there  is  no  distinct  capsule,  no  evident  budding, 
no  mycelium.  Moreover,  the  staining  results  apparently  indicate 
that  they  are  a  degeneration  product  of  nuclear  substance.  It 
was  hoped  that  the  organism  as  it  appeared  in  animal  would 
afford  a  clue  to  its  appearance  in  human  tissues.  The  kidney 
lesions  in  mice  are  very  clear.  Virtually  they  consist  of  a  mass  of 
delicate,  short  or  long,  segmented  fungous  filaments,  and  small 
round  forms,  which  stain  well  with  methylene  blue.  Similar 
forms  could  not  be  found  in  the  human  tissue. 

The  idea  occurred  that  by  inoculating  the  organism  subcuta- 
neously  into  a  guinea-pig  at  several  points  and  resecting  specimens 
from  all  stages  of  the  process — incipient  induration,  abscess  for- 
mation and  resolution — perhaps  a  means  would  be  suggested  of 
recognizing  the  organism  in  the  lip  ''tumor."  Details  are  cited 
imder  animal  experiments.  The  experiment  did  not  yield  the 
needed  help.  The  first  piece  of  tissue  resected,  however,  afforded 
a  suggestion.     It  appeared  grossly  as  a  solid  cellular  mass,  resem- 


66  Contributions  to  Medical  Science 

bling  closely  a  cross-section  of  the  lip  tissue.  It  required  the 
closest  search  to  find  a  few  scattered  spherical  organisms  with 
typical  structure,  capsule,  and  all.  Yet  in  a  few  days  this  same 
area  broke  down  into  a  thick,  yellowish  pus,  which  was  loaded 
with  mycelium  and  spherical  forms.  This  points  very  strongly  to 
an  intermediate  unrecognized  form,  which  may  exist  in  tissue. 

Sections  of  the  human  tissue  were  treated  with  potassium 
hydrate  solutions  and  digested  with  pepsin,  but  organisms  were 
not  revealed. 


Animal  Experiments. —  Guinea-pigs.  i.  An  intraperitoneal  inoculation  with 
teased  tissue.  Death  two  months  later,  when  a  number  of  animals  died  from  some 
obscure  cause.     Postmortem  findings  and  cultures  negative. 

2.  Teased  tissue  was  placed  subcutaneously  in  the  right  inguinal  region;  also 
the  abdominal  skin  was  scarified  and  portions  of  tissue  were  rubbed  into  the  lesion. 
Five  days  later  a  sinus  formed  at  the  point  of  the  subcutaneous  inoculation,  which 
discharged  cheesy  pus  for  a  week  and  then  healed.  Neighboring  lymph  glands 
enlarged,  and  a  temperature  of  104°  F.  developed,  which  soon  disappeared.  The  pus, 
mounted  in  KOH  solution,  showed  both  the  spherical  and  myceUal  forms  of  the 
organism.  Cultures  yielded  besides  the  fungus  a  streptococcus,  staphylococci,  and  a 
long  baciUus.     The  scarification  wound  healed  promptly. 

3.  One  c.c.  of  a  ten-day-old  bouillon  culture  injected  subcutaneously  in  the 
interscapular  region.  Temperature  of  101°  F.  resulted.  An  induration  formed, 
which  subsided  after  five  days  without  suppurating.  The  animal  died  from  unknown 
causes,  in  company  with  guinea-pig  i.  The  postmortem  findings  and  cultures  from 
organs  were  negative. 

4.  Twenty  minims  of  a  si.x-day  glucose  bouillon  culture  inoculated  subcutaneously 
in  the  interscapular  region.  A  moderate  fever  resulted,  which  subsided  in  three  or 
four  days.  Pus  formed  in  two  days.  It  contained  grotesque  mycehal  fragments  and 
single  adult  and  budding  forms.  Cultures  yielded  the  fungus.  Later  three  sub- 
cutaneous inoculations  from  glucose  bouillon  cultures  were  made  in  this  animal 
simultaneously.     The  resulting  lesions  were  resected  at  different  stages. 

First.  A  deep,  indurated  mass,  which  developed  in  five  daj^s  after  inoculation- 
On  section  it  appeared  homogeneous  and  grayish-white,  rather  anemic.  Pus  had  not 
formed.  Sections  showed  a  central  mass  of  leukocytes,  erythrocytes,  and  connective- 
tissue  cells,  with  occasional  fragments  of  elastic  tissue.  Curious  forms  of  phagocytosis 
or  cell  invasion  were  conspicuous.  After  much  search,  spherical  and  budding  forms 
of  the  fungus  were  found  in  small  numbers,  but  there  was  no  mycelium. 

The  presence  of  peripherally  disposed  granules,  which  stained  well,  had  not 
been  seen  previously  in  the  organism.  Otherwise  the  structure  corresponded  to 
previous  descriptions. 

Second.  A  suppurating  lesion  ten  days  old.  The  thick,  whitish  pus  contained, 
in  addition  to  spherical  and  budding  forms,  grotesque  and  imperfect  mycelial  frag- 
ments. In  stained  sections  it  was  impossible  to  recognize  anything  but  leukocytes, 
connective-tissue  cells,  fibrin,  and  debris  in  the  abscess  wall. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  67 

The  explanation  is  not  clear  as  to  why  the  organism  should  be  found  in  abundance 
in  the  pus  and  yet  be  unrecognizable  in  the  stained  section  of  the  abscess  wall. 

Third.  The  third  focus  discharged  pus,  and  when  this  had  nearly  ceased  the 
lesion  was  excised.  Buried  in  the  subcutaneous  tissue  was  a  mass  composed  of  poly- 
morphonuclear leukocytes,  endothelioid  cells,  phagocytic  endothelioid  and  giant 
cells,  degenerating  red  blood  cells,  nuclear  and  granular  detritus,  and  fibrin.  The 
phagocytic  endothelioid  cells  were  sometimes  multinuclear,  without  presenting  the 
appearance  of  giant  cells.  There  was  no  characteristic  arrangement  of  the  nuclei, 
nor  was  the  protoplasm  granular.  They  contained  polymorphonuclear  leukocytes, 
other  endothelioid  cells,  relatively  minute  spherical  bodies  staining  intensely  with 
hematoxylin  and  vacuoles.  The  minute  spherical  bodies  are  considered  remnants 
of  nuclear  tissue,  though  the  possibility  is  in  mind  that  they  may  be  small  forms  of 
the  organism  inoculated.  Many  are  found  extracellular  and  imbedded  in  a  granular 
matrix.  A  capsule  could  not  be  demonstrated,  and  there  was  nothing  fungoid  in 
their  arrangement. 

Rabbits,  i.  Subcutaneous  inoculation  of  fresh  tissue.  Death  after  four  days. 
Cultures  yielded  only  Staphylococcus  pyogenes  albus. 

2.  One  c.c,  of  a  ten-day  bouillon  culture  inoculated  into  vein  of  the  right  ear. 
The  culture  had  been  kept  at  the  room  temperature  and  contained  abundant  mycehum, 
budding  cells,  and  a  few  degenerate  forms.  The  temperature  rose  to  105.5°  F.  on 
the  second  day,  returning  to  normal  after  four  or  five  days.  On  the  second  day  an 
induration  3  cm.  long  and  i  cm.  broad  formed  in  the  course  of  the  vein,  beginning 
at  the  point  of  inoculation  and  extending  centrally.  After  twelve  days  pus  was 
discharged,  in  which  degenerate  forms  of  the  organism  were  found.  Cultures  were 
negative. 

Mice.^  I.  One-half  c.c.  of  a  three-day-old  glucose  bouillon  culture  inoculated 
subcutaneously.  Died  in  thirty  hours.  Cultures  of  the  fungus  obtained  from  the 
kidneys. 

2.  A  portion  of  a  glycerin  agar  culture  was  suspended  in  bouillon  and  twenty 
minims  of  the  mixture  inoculated  subcutaneously.  The  animal  appeared  sick  for 
a  few  days,  but  recovered. 

Later,  ten  minims  of  a  six-day-old  glucose  bouillon  culture  were  inoculated.  On 
the  second  day  the  animal  died.  Pure  cultures  of  the  organism  were  obtained  from 
the  blood  and  kidneys. 

3.  Portion  of  culture  from  a  solid  medium  was  rubbed  into  a  scarification  of  the 
skin  over  the  buttock.  The  animal  died  in  two  days.  Postmortem,  microscopic, 
and  bacteriological  examination  negative. 

4.  Twelve  minims  of  a  twelve-day-old  glucose  bouillon  culture  inoculated  subcu- 
taneously. Died  on  the  second  day.  Organism  recovered  in  pure  culture  from 
kidneys,  blood,  and  spleen. 

Tissue  changes  in  mice. — Except  an  unusual  vascularity  of  the 
kidneys,  gross  changes  were  not  noted.  All  organs  were  fixed  in 
alcohol  and  imbedded  in  paraffin. 

Kidneys. — Acute  granular  degeneration  of  the  tubular  epithe- 
lium is  present  in  all  cases,  but  in  varying  degrees  of  intensity.     In 

'  Ordinary  house  mice  were  used. 


68  Contributions  to  Medical  Science 

mouse  2  well-staining  nuclei  are  rare.  In  mouse  i  this  degeneration 
is  much  less  marked.  The  chief  interest  centers  around  tubules  and 
groups  of  tubules,  which  are  unstained  or  faintly  stained  with 
hematoxyhn,  but  take  up  methylene  blue  deeply.  They  are 
granular  masses  and  contain  small  spherules  and  short  threads, 
which  may  extend  into  the  interstitial  tissue.  Often  what  appear 
to  be  spherules  are  really  cross-sections  of  threads.  With  a 
methylene  blue  stain,  and  an  oil-immersion  objective,  the  spherules 
and  threads  are  recognized  as  part  of  a  micro-organism.  Larger 
spherical  capsulated  organisms  are  sometimes  seen  in  the  central 
part  of  an  area,  which  correspond  to  the  adult  spherical  cells  s.een 
in  cultures.     The  protoplasm  of  such  cells  often  stains  dimly. 

The  predominating  form  is  a  segmented  thread,  not  unlike 
bacilH  in  chain  form,  except  for  the  large  size.  The  segments  are 
from  one  and  one-half  to  four  microns  wide,  and  the  length  from 
two  to  four  times  greater  than  the  breadth.  The  ends  are  rounded 
bluntly.  Not  more  than  six  segments  have  been  seen  in  a  thread, 
and  the  longer  chains  taper  to  a  narrower  extremity.  Often  the 
terminal,  and  occasionally  an  intermediate,  segment  is  oval  in 
form.  Isolated  oval  forms  are  common,  but  the  spherical  capsu- 
lated organism  is  seen  less  frequently.  The  latter  are  constantly 
larger  than  the  former.  Branching  threads  are  not  observed. 
With  polychrome  methylene  blue,  the  periphery  of  both  mycelium 
and  oval  forms  stains  deeply,  leaving  an  unstained  or  faintly 
stained  center.  The  large  spherical  forms  present  a  thin,  sharply 
stained  periphery,  which  apparently  represents  the  capsule.  The 
earhest  condition  found  is  that  of  a  few  organisms  in  the  lumen  of 
a  tubule,  the  epitheUal  cells  being  very  granular.  In  the  larger 
foci  no  traces  of  kidney  cells  are  apparent,  the  areas  consisting  of 
dense  accumulations  of  the  organism.  There  is  virtually  no  inter- 
stitial proliferation,  and  a  marked  absence  of  leukocytes.  No 
plasma-  nor  mast-cells  are  found ;  only  a  thin  shell  of  cells  surrounds 
a  focus,  and  these  are  of  the  endothehoid  and  lymphoid  type. 

Heart. — Circumscribed  areas  of  infiltration,  separating  and 
occasionally  destroying  the  muscle  fibers,  are  widespread.  In 
mouse  I  no  organism  was  demonstrated  positively  in  these  cell 
masses.     In   the   remaining  mice,   adult   and  budding  spherical 


Oidiomycosis  of  the  Skin  and  Its  Fungi  69 

forms  were  numerous.  Single  adult  forms  measured  from  six 
to  ten  microns  in  diameter.  No  mycelium  was  found,  though 
elongated  single  cells  were  fairly  numerous. 

Lungs. — Only  extreme  congestion.  The  liver  and  spleen 
normal. 

White  rat.  Twenty  minims  of  a  four-day-old  glucose  bouillon  culture  inoculated 
intraperitoneally  on  November  27,  1900.  No  symptoms  followed,  and  the  animal 
is  still  living. 

Dogs.  I.  On  October  27,  1900,  four  cubic  centimeters  of  a  four-day  glucose 
bouillon  culture  were  injected  into  the  left  saphenous  vein.  Prostration,  fever,  and 
emaciation  followed.     After  a  week  gradual  recovery  set  in. 

On  November  28,  1900,  four  cubic  centimeters  of  a  one-month-old  glucose  bouillon 
culture  were  injected  into  the  right  axillary  vein.  Symptoms  like  those  following 
the  first  inoculation  developed,  but  of  less  severity.  On  December  3,  1900,  while 
still  sick,  the  animal  was  chloroformed.  The  axillary  glands  on  the  right  side  were 
very  large,  and  there  was  mild  general  lymphadenopathy.  They  presented  no  soften- 
ing. No  gross  changes  in  heart,  liver,  and  lungs.  Spleen  very  hard  and  malpighian 
corpuscles  are  large  and  are  shelled  out  easily.  Kidneys  are  hard,  and  there  are  many 
subcapsular,  white,  scar-like  points. 

Smears  from  the  wound,  which  had  been  torn  open  by  the  animal,  showed  many 
budding  fungus  cells.  None  were  found  in  smears  from  the  viscera  and  the  heart's 
blood. 

Inoculations  from  the  heart's  blood,  lungs,  liver,  kidneys,  and  spleen  produced 
no  growths  on  suitable  media. 

Microscopic  examination  of  an  axillary  lymph  gland  showed  new-formed  connect- 
ive tissue  and  large  masses  of  plasma  cells.  There  were  intracellular  hyalin  bodies, 
but  no  fungous  cells  were  recognizable.  The  liver  contained  small  areas  of  round 
cell  infiltration,  but  the  organism  could  not  be  detected.  In  the  lung  were  small 
masses  of  new  fibrous  tissue,  but  no  fungi.  The  white  subcapsular  areas  in  the  kidneys 
were  composed  of  masses  of  plasma  cells,  a  few  endothelioid  cells,  and  portions  of 
kidney  tubules.  Other  plasma  cell  foci  were  scattered  throughout  the  kidney,  but 
no  organism  could  be  recognized. 

The  heart  and  spleen  appeared  normal. 

2.  Subcutaneous  inoculation  of  two  cubic  centimeters  of  a  three-day-old  plain 
bouillon  culture.  An  induration  developed,  which  subsided  in  five  days.  The  autopsy, 
ten  days  later,  showed  no  changes.  Cultures  from  the  viscera  were  sterile,  and 
microscopically  the  organs  were  normal. 

Human.  An  area  on  the  calf  one-half  inch  square  was  scarified  superficially  and 
a  portion  of  a  growth  on  an  agar  slant  thoroughly  rubbed  in.  Soreness  for  three  or 
four  days  resulted,  when  a  healthy  crust  formed  and  perfect  healing  occurred. 

Morphology,  etc. — The  organism  may  proliferate  by  (i) 
budding  of  spherical  or  oval  cells;  (2)  the  formation  of  lateral  and 
terminal  conidia;  and  (3)  by  the  splitting-up  of  the  segmented 
mycelium  into  chains  of  "spores," 


yo  Contributions  to  Medical  Science 

The  formation  of  endospores  and  the  presence  of  a  nucleus  have 
been  sought  under  many  conditions.  The  protoplasm  possesses 
inconstant  morphological  elements.  It  may  be  absolutely  clear, 
structureless,  or  may  undergo  differentiation  into  apparently 
complex  elements.  In  small  buds  and  young  isolated  cells  it  is 
usually  clear,  and  with  all  dyes  stains  evenly  and  deeply.  In 
active  full-grown  cells  it  may  possess  one  or  more  of  the  following 
differentiated  elements:  (i)  granules,  (2)  vacuoles,  and  (3)  a 
nucleus-like  or  spore-like  (?)  structure;  or  it  may  be  absolutely 
clear  and  stain  like  the  younger  cells.  There  may  be  only  one 
granule  or  several.  In  living  cells  some  of  them  move  actively 
in  a  vibrating  manner  through  the  protoplasm.  Others  are  quies- 
cent. In  stained  mounts  perhaps  only  one  or  two  granules  will 
take  up  the  dye;  or  a  small  granular  stained  mass  may  lie  in  the 
center  or  at  one  side  of  the  cell.  It  is  common  to  find  a  deeply 
stained,  irregular,  semi-lunar  or  horse-shoe  shaped  mass,  closely 
appHed  to  the  cell  wall,  occupying  one-half  to  three-fourths  of  the 
periphery.  As  these  granules  are  not  found  in  the  buds,  their 
relation  to  proliferation,  if  they  have  any,  must  be  an  indirect 
one. 

The  nucleus-  or  spore-like  structure  does  not  occur  in  all  active 
adult  cells.  It  is  often,  but  not  uniformly,  present  in  the  parent 
budding  cell.  When  the  cell  is  not  budding,  this  structure  usually 
lies  in  the  center  of  the  protoplasm.  In  oval  cells  it  Hes  in  the 
smaller  pole.  As  budding  begins,  and  during  the  process,  it 
approximates  the  cell  wall  where  the  bud  is  to  arise,  and  a  similar 
body  may  or  may  not  appear  in  the  daughter  cell.  It  may  contain 
one  or  two  highly  refractive  granules.  The  conditions  of  its 
appearance  and  disappearance  have  not  been  made  out.  The 
relation  it  bears  to  reproduction  is  not  clear,  as  cells  bud  in  which 
this  substance  is  not  seen.  That  it  is  a  nucleus  in  the  ordinary 
sense  of  the  word  cannot  be  accepted,  as  it  is  inconstant,  pos- 
sesses no  nuclear  membrane,  does  not  participate  directly  in  cell 
division,  and  is  not  stained  constantly  with  nuclear  dyes.  In 
using  hematoxylin,  very  dilute  carbol-fuchsin,  and  neutral  red  as 
vital  stains,  it  colors  more  quickly  and  more  densely  than  other 
structures;  but  in  fixed  films  it  could  not  be  clearly  differentiated 


Oidiomycosis  of  the  Skin  and  Its  Fungi  71 

by  staining  methods.  That  it  is  a  spore  is  also  uncertain.  It  has 
not  been  observed  to  be  hberated  and  to  develop  new  cells;  and 
it  does  not  take  part  in  a  "free  cell-formation,"  and  lacks  the 
structure  demanded  of  spores.  It  is  often,  but  not  uniformly, 
present  in  mycelium.  It  occurs  both  in  the  segmented  and  the 
unsegmented  threads,  but  is  more  common  in  the  former,  where 
they  may  have  a  chain-like  disposition,  each  element  being  separated 
from  its  neighbor  by  the  surrounding  clear  substance;  or  they 
may  be  irregularly  distributed  and  assume  different  sizes.  In 
general,  this  structure  is  present  in  actively  proliferating  cells 
(mycelium  or  budding  cells),  and  absent  in  old,  much  vacuolated, 
involution  forms. 

Vacuoles  are  found  most  of  all  in  the  large  old  involution  forms, 
less  often  in  the  active  adult  cells,  and  almost  never  in  young 
cells  or  buds.  In  involution  cells  they  are  often  multiple,  and 
occupy  nearly  all  the  intracapsular  space,  being  surrounded  only 
by  a  thin  zone  of  granular  protoplasm.  Other  old  forms  show 
absolutely  no  internal  structure,  only  a  hollow  capsule  being 
visible  either  in  living  or  stained  specimens. 

It  was  endeavored  to  produce  endogenous  spores  by  growth  in 
distilled  water,  at  low  temperatures,  high  temperatures  (41°  C), 
on  highly  acid  media,  in  potassium  iodide  solutions,  and  on  a 
moist  gypsum  block.  Very  highly  refractive  globules,  one  to 
four  in  a  cell,  which  sometimes  resulted,^  were  considered  plasmo- 
lytic  effects. 

In  a  hanging  drop  of  dog's  blood-serum,  an  immediate  formation 
of  mycelium  begins.  A  spherical  cell  sends  out  a  small  filamentous 
shoot,  which  increases  in  length  rapidly,  becomes  segmented,  and 
acquires  in  irregular  points  a  mildly  refractive  spherical  or  oval 
body,  tinted  a  faint  pink  or  orange.  This  is  the  nucleus-like 
structure  spoken  of,  and  is  also  formed  in  the  spherical  cells  giving 
rise  to  the  threads  (Fig.  14,  Plate  7).  The  mixture  of  spherical 
cells  and  mycelium  is  similar  to  that  formed  by  the  microsporon 
furfur. 

Resume. — The  reasons  for  considering  the  fungus  described  the 
cause  of  the  disease  in  this  case  are  as  follows: 

I .  The  chnical  history  suggests  an  unusual  etiology. 


72  Contributions  to  Medical  Science 

2.  Voluminous  cultures  of  the  fungus  were  obtained  from  the 
deep  portion  of  the  "tumor"  under  aseptic  precautions. 

3.  The  histopathology  of  the  tissue — intra-epitheHal  abscesses, 
epithehal  hyperplasia,  and  the  character  of  infiltrating  cells — places 
it  with  a  group  of  diseases  which  have  been  proven  to  be  caused  by 
fungi  similar  to  the  one  under  consideration. 

4.  Portions  of  the  tumor,  introduced  subcutaneously  into 
guinea-pigs,  produced  an  abscess  in  which  was  found  the  yeast 
obtained  in  cultures  from  the  tissues. 

5.  Its  variable  morphology  in  animal  tissues  gives  ground  for 
the  belief  that  in  human  tissues  it  may  have  assumed  a  form 
different  from  that  observed  in  animals  or  cultures,  and  hence 
Ukely  to  escape  detection  by  the  means  used.  As  an  example  of 
this  variability :  In  mice  myceUal  forms  were  found  in  the  kidneys 
and  an  oval  and  spherical  form  in  the  heart.  In  subcutaneous 
inoculation  in  guinea-pigs  it  was  almost  impossible  to  find  the 
fungus  in  the  densely  cellular  masses  which  immediately  preceded 
pus  formation,  although  quantities  had  been  injected;  yet  a  day 
or  two  later,  when  hquef  action  had  occurred,  there  was  no  difficulty 
in  demonstrating  large  numbers  of  the  organism,  principally  as 
hyphae.  Previous  to  liquefaction,  the  forms  found  were  of  the 
oval  type. 

Case  //.—Clinical  history  (Dr.  Montgomery).— T.  R.,  aged 
thirty-eight,  married,  by  occupation  a  carpenter. 

On  June  15,  1894,  while  tearing  down  an  old  kitchen,  he  injured 
the  back  of  his  hand.  The  place  became  covered  with  a  crust, 
on  the  removal  of  which,  several  days  later,  he  saw  an  elevated, 
angry-looking  spot.  Before  the  hand  was  quite  well,  a  barber  cut 
a  wart  from  his  neck,  just  under  the  chin.  In  a  few  days  there 
appeared  at  this  point  a  little  elevation,  covered  by  a  crust. 

The  clinical  picture  was  largely  that  of  a  verrucous  tuberculosis, 
though  in  places  it  suggested  more  a  papillary  epithelioma.  The 
border  was  elevated  from  a  sixteenth  to  a  quarter  of  an  inch, 
and  was  sharply  defined,  except  for  a  narrow  zone  of  bluish-red 
congestion,  which  for  the  greater  part  was  inconspicuous.  The 
growth  for  the  most  part  was  verrucous,  situated  on  a  very  slightly 


Oidiomycosis  of  the  Skin  and  Its  Fungi  73 

infiltrated  base,  though  in  places  the  latter  was  quite  firmly  indu- 
rated. Here  and  there  the  base  was  quite  soft,  and  pus  could  be 
pressed  out  between  the  projecting  papillae. 

The  pus  was  examined  under  the  microscope  and  a  nmnber  of 
culture  media  were  inoculated.  Tissue  was  removed  from  one 
border.  The  major  portion  of  it  was  hardened  in  alcohol,  but  a 
small  piece  was  teased  in  distilled  water  and  used  for  the  purpose 
of  inoculating  more  media.  Bits  of  this  tissue  were  introduced 
into  the  subcutaneous  tissue  of  two  guinea-pigs  and  a  rabbit.  On 
the  culture  media  no  growths  were  obtained  except  those  with  pus 
organisms,  while  the  results  of  the  animal  inoculations  were  abso- 
lutely negative.  Microscopical  examinations  of  the  sections 
showed  an  unusual  appearance,  which  will  be  described  later.  The 
diagnosis  made  at  this  time  was  that  of  a  probable  cutaneous 
tuberculosis,  though  the  possibility  of  blastomycetic  infection  was 
considered.  DiUgent  examination  of  the  stained  specimens 
failed  to  show  blastomycetes. 

At  the  meeting  of  the  American  Dermatological  Association  in 
1898,  I  mentioned  this  case  during  the  discussion  of  Dr.  Shepherd's 
paper  on  "A  Strange  Case  of  Granuloma  of  the  Face  and  Extremi- 
ties," the  photograph  of  his  case  and  his  description  bearing  a 
striking  resemblance  to  the  clinical  features  presented  by  this 
case  a  few  weeks  previously. 

The  man  did  not  present  himself  again  until  August  26,  1899, 
eighteen  months  after  his  first  visit.  The  disease  had  extended 
over  the  left  cheek  to  the  nose,  had  surrounded  the  left  orbit  and 
involved  both  upper  and  lower  eyelids;  the  cicatricial  tissue 
producing  aversion  of  the  lower  lid. 

In  places  there  are  raised,  smoother  areas;  in  others  the  base 
beneath  the  papillae  is  soft,  and  pus  can  be  expressed.  Various 
culture  media  were  inoculated  with  the  pus  and  with  tissue  taken 
from  an  advancing  border.  No  cultures  of  blastomyces  were 
obtained,  though  the  organisms  were  demonstrated  later  in  typical 
forms  in  pus  and  tissue  which  had  been  subjected  to  the  action 
of  a  weak  solution  of  potassium  hydrate. 

The  man  again  disappeared  from  view  for  four  months,  at  the 
end  of  which  time  he  applied  for  admission  to  the  Illinois  Eye  and 


74  Contributions  to  Medical  Science 

Ear  Infirmary.  To  Dr.  Dodd  of  that  institution,  who  came  to 
consult  me  regarding  the  man,  I  am  indebted  for  further  oppor- 
tunity to  study  the  case,  and  for  carrying  out  treatment  with  the 
iodide  of  potassium,  imder  the  influence  of  which,  in  large  doses, 
the  progress  of  the  disease  has  been  arrested,  and  the  verrucous 
growth  is  being  replaced  by  the  characteristic  smooth  red  scar. 

Fresh  Tissue. — Tissue  was  taken  for  cultures  February  8, 
1900.  A  portion  of  this  tissue,  teased  and  mounted  in  25  per  cent 
potassium  hydroxide  solution,  shows  typical  blastomycetes 
proliferating  by  budding.  These  forms  measure  about  twelve 
microns  in  diameter. 

Besides  the  large  cells,  another  form  is  seen,  which  averages 
3.75  microns  in  diameter.  They  are  recognized  as  blastomycetes 
or  as  a  similar  organism  because  of  a  distinct  capsule,  a  central 
protoplasm,  and  proliferation  by  budding.  The  capsule  is  deUcate 
and  structureless.  Very  fine  granules  are  seen  in  the  protoplasm, 
and  no  nuclei  or  vacuoles  are  observable.  The  budding  process 
is  identical  with  that  seen  in  the  larger  forms.  They  exhibit  a 
marked  tendency  to  form  in  chains,  some  of  the  segments  being 
oblong  or  cylindrical,  imitating  the  segments  of  mycelial  threads. 
Chains  containing  from  four  to  six  segments  were  observed  com- 
monly, none,  however,  possessing  more  than  eight.  These  forms 
were  observed  in  a  subsequent  examination  of  teased  tissue,  at 
no  time  being  seen  in  the  pus  of  the  miliary  abscesses. 

Cultures. — On  August  26, 1899,  culture  media  of  potato,  blood- 
serum,  and  glucose  agar  were  inoculated  both  from  teased  tissue 
and  from  the  pus  obtained  from  miliary  abscesses.  The  results 
were  negative.  The  diflferent  staphylococci  only  were  obtained. 
On  February  8,  1900,  four  plain  agar,  four  glycerin  agar,  and  two 
plain  bouillon  tubes  were  inoculated  with  verrucous  tissue,  which 
had  previously  been  teased  finely  in  bouillon.  One  half  of  these 
tubes  were  kept  in  the  incubator  and  the  other  half  at  room  tem- 
perature. Very  profuse  growths  of  staphylococci,  a  few  colonies 
of  streptococci  and  saprophytic  bacilli  were  the  result  of  these 
inoculations.  The  tubes  were  kept  under  observation  for  two 
months  and  no  growth  of  blastomycetes  developed. 

Animal  Experiments. —  On  August  26,  1899,  a  guinea-pig  was  inoculated  intra- 
peritoneally  with  a  portion  of  the  verrucous  tissue,  and  at  the  same  time  the  abdomen 


Oidiomycosis  of  the  Skin  and  Its  Fungi  75 

at  one  point  was  scarified  and  bits  of  verrucous  tissue  rubbed  thoroughly  into  the 
lesion.  This  animal  died  in  two  days.  The  autopsy  showed  peritonitis,  and  cultures 
from  the  peritoneum,  viscera,  and  the  blood  resulted  in  a  growth  of  staphylococci. 
A  rabbit  on  the  same  day  was  inoculated  subcutaneously  with  a  portion  of  the  teased 
tissue.  A  mild  reactive  inflammation  was  the  result  at  the  seat  of  inoculation.  This 
induration  subsided  gradually  without  the  formation  of  pus.  Cultures  of  the  indurated 
nodule  gave  no  results.  On  the  same  day  a  white  mouse  was  inoculated  with  a  piece 
of  teased  tissue  beneath  the  skin  of  the  back.  The  mouse  died  in  five  days  of 
staphylococcus  septicemia. 

HiSTOPATHOLOGY. — For  histological  purposes,  two  portions  of 
tissue  were  removed  from  the  periphery  of  the  diseased  area,  each 
containing  a  portion  of  adjacent  healthy  skin.  The  tissues  were 
hardened  and  fixed  in  absolute  alcohol,  and  imbedded  in  celloidin. 
The  microscopic  pictures  of  the  two  pieces  of  tissue  are  practically 
identical.  Sections  were  stained  with  hematoxylin  and  eosin, 
with  polychrome  methylene  blue,  Gram-Weigert  method,  acid 
orcein,  and  with  carbol-fuchsin  for  tubercle  bacilli.  The  surfaces 
of  the  sections  were  covered  with  accumulations  of  loosened  horny 
material  and  red  blood  corpuscles.  Gram's  method  showed  many 
cocci  among  the  loosened  homy  lamellae,  and  some  buried  more 
deeply  in  the  horny  layer.  The  hair  follicles  were  accentuated 
in  their  width.  There  was  a  marked  irregularity  of  the  surface, 
the  homy  layer  in  places  dipping  down  deeply,  and  in  these  depres- 
sions occurred  masses  of  homy  lamellae.  In  the  diseased  portion, 
the  horny  cells  showed  the  remains  of  nuclei.  The  deeper  horny 
cells  had  nuclei  uniformly.  The  stratum  lucidum  was  not  demon- 
strable in  the  diseased  portion.  The  stratum  granulosum,  however, 
was  very  conspicuous,  except  where  abscesses  actually  communi- 
cated with  the  surface.  It  averaged  from  three  to  six  cells  deep. 
The  rete  showed  extensive  proliferation,  long,  coarse,  finger-like 
projections  dipping  down  deeply  into  the  cutis,  almost  to  the 
subcutaneous  tissue.  These  processes  were  greatly  deformed,  at 
one  place  being  very  delicate,  constricted,  and  almost  separated 
from  the  surface  rete,  and  immediately  below  perhaps  enormously 
enlarged  from  accumulations  of  leukocytes.  A  columnar  basal 
layer  was  quite  well  maintained  throughout  all  the  gyrations.  The 
individual  rete  cells  possessed  deeply  staining  nuclei  and  granular 
protoplasm,  which  took  up  eosin  rather  intensely.  The  prickles 
stood  out  prominently.     Many  of  the  nuclei  were  shrunken,  allow- 


76  Contributions  to  Medical  Science 

ing  the  formation  of  a  clear  space  between  the  nucleus  and  the  cell 
protoplasm.  In  certain  portions  small  foci  of  rete  cells  appeared 
necrotic.  The  sizes  of  the  individual  cells  were  subject  to  consider- 
able variation.  The  deeper  cells  were  quite  large,  conspicuously 
granular,  and  the  intercellular  spaces  widely  dilated.  Many 
partial  whorl  formations  were  seen.  These  whorls  usually  did  not 
contain  a  demonstrable  hornified  center.  Infiltrating  cells,  largely 
polymorphonuclear  leukocytes,  were  very  abundant  in  the  inter- 
epithehal  spaces,  and  in  addition  there  were  a  relatively  small 
number  of  mononuclear  leukocytes,  and  occasional  eosinophiles. 
In  some  places  the  leukocytes  accumulated  to  form  small  abscesses. 
The  rete  cells  immediately  surrounding  the  abscesses  have  been 
compressed  and  flattened,  and  partially  hornified.  This  went  so 
far  that  the  surrounding  cells  gave  the  appearance  of  a  limiting 
abscess  membrane,  composed  of  fiber-like  lamellae,  the  cells  of 
which  largely  retained  their  nuclei. 

Cutis. — The  papillary  layer  had  lost  its  typical  structure  and 
form.  There  were  dense  accumulations  of  small  cells,  and  the 
corium  was  exceedingly  vascular  from  dilated  and  newly  formed 
blood-vessels,  lymph  channels,  and  spaces.  The  fibrous  and 
elastic  tissues  were  broken  up  and  separated  by  infiltrating  cells. 
In  many  places  the  accumulation  of  leukocytes  was  so  dense  that 
all  normal  tissue  was  either  replaced  or  obscured.  The  infiltration 
extended  as  far  as  the  subcutaneous  fibrous  and  fatty  tissue;  and 
even  into  these,  columns  of  infiltrating  cells  follow  the  blood-vessels. 
The  stroma  of  the  fatty  tissue  was  infiltrated  in  places,  and  the 
fixed  cells  were  in  a  state  of  prohferation.  The  infiltrating  cells 
were  polymorpho-  and  mono-nuclear  leukocytes,  plasma  cells, 
and  very  few  eosinophiles;  also  a  relatively  large  number  of  mast- 
cells  and  of  newly  formed  connective-tissue  cells.  Of  these  the 
polymorphonuclear  leukocytes  formed  by  far  the  greatest  number. 
They  were  somewhat  irregularly  distributed,  in  one  place  being 
so  densely  crowded  as  to  constitute  small  abscesses,  in  others  less 
densely  and  evenly  distributed.  The  plasma  cells  were  unevenly 
distributed,  their  tendency  being  to  shun  the  foci  of  most  acute 
inflammation.  They  were  collected  in  masses  at  a  short  distance 
from  the  abscesses,  however;  in  general,  where  the  process  was 


Oidiomycosis  of  the  Skin  and  Its  Fungi  77 

subacute.  There  was  nothing  peculiar  about  the  plasma  cells 
in  this  case  to  distinguish  them  from  the  classical  cell  of  that  name. 
They  were  not  gathered  in  masses  to  the  exclusion  of  other  cells, 
leukocytes,  mast-cells,  or  connective-tissue  cells,  but  they  did 
occur  in  loosely  constructed  masses,  mostly  near  the  blood-vessels; 
hence  certain  sections  showed  these  cells  in  the  formation  of  columns. 

Giant  cells  were  numerous  and  occurred  in  noticeable  groups. 

The  miliary  abscesses. — In  the  intra-epitheHal  abscesses  were, 
besides  the  leukocytes,  loosened  epithehal  cells,  the  forms  of  which 
vary  greatly;  a  certain  amount  of  nondescript  debris,  fibrin,  and 
the  organism.  There  were  occasional  plasma  cells.  For  the  most 
part  the  nuclei  of  all  the  cells  stained  distinctly,  the  number  of 
broken-up  cells  being  small.  The  parasites  were  circular  or 
sHghtly  oblong,  existing  singly,  in  pairs,  or  in  clumps  of  three  or 
four.  Typical  budding  was  common.  The  individual  spherical 
form  consisted  of  a  doubly  contoured  capsule,  which  stained 
inconstantly  with  methylene  blue,  and  a  central  protoplasm 
staining  a  lighter  blue  and  separated  from  the  capsule  by  a  con- 
centric relatively  clear  zone.  When  they  occurred  in  pairs,  there 
were  certain  differences  between  the  elements  making  up  the  pair. 
One  of  the  cells  may  have  a  thicker,  more  deeply  staining  capsule 
than  the  other,  and  a  larger  area  of  protoplasm.  Distinctly 
vacuolated  forms  were  seen  very  rarely  in  this  tissue.  A  condition 
of  interest  existing  in  many  cells  is  that  of  large  granules  arranged 
peripherally  in  the  protoplasm  of  the  fungous  cell.  The  number 
of  these  in  different  cells  was  subject  to  considerable  variation. 
As  many  as  twenty-four  were  seen  in  a  single  cell,  the  usual  num- 
ber being  eight  or  ten. 

The  budding  seen  in  this  case  differed  in  no  way  from  that 
described  by  previous  observers.  The  number  of  organisms  was 
relatively  few  and  they  were  seen  only  occasionally  outside  of 
true  abscesses.  They  were  not  seen  within  tissue  cells.  A  few 
were  found  in  the  granulation  tissue  of  the  cuts. 

Case  1 11.^ — CHnical  history. — Mr.  F.  S.  S.,  age  33  years,  by 
occupation  a  farmer,  resident  of  Elburn,  111.,  applied  for  treatment 

» A  brief  report  of  Cases  III  and  IV  has  been  made  by  Hyde  and  Ricketts,  Jour.  Cutaneous  and 
Genito-Urinary  Diseases,  1901. 


78  Contributions  to  Medical  Science 

December  15,  1899.  His  family  history  was  satisfactory.  For 
three  years  previous  to  the  present  date  he  had  been  engaged 
every  autumn  in  handHng  a  threshing-machine.  His  general 
appearance  was  that  of  a  man  in  sound  health  and  with  good 
habits. 

The  present  cutaneous  affection  began  about  two  years  ago. 
When  examined,  the  region  involved  was  recognized  to  be  the  left 
lower  lid  and  the  parts  adjacent  in  both  cheek  and  temple. 

Cultures. — Mounted  in  30  per  cent  KOH  solution,  pus  from 
the  miliary  abscesses  shows  a  multitude  of  ordinary  pus  cells, 
which  soon  disintegrate,  and  in  addition  cells  of  another  nature, 
which  resist  the  action  of  the  alkaH.  The  latter  are  spherical  in 
form  and  arranged  singly,  in  pairs,  or,  more  rarely,  in  groups  of 
three  or  four,  the  individual  cells  being  united.  Fully  developed 
cells  have  an  average  diameter  of  twelve  microns.  The  structural 
features  are:  an  external,  highly  refractive,  homogeneous,  doubly 
contoured  capsule,  having  a  thickness  of  from  one-fourth  to  one 
micron;  a  "clear  zone"  internal  to  the  capsule,  presenting  no 
structure  and  not  constant;  and,  finally,  a  central  protoplasmic 
substance,  which  is  sharply  separated  from  the  clear  space  by  a 
delicate  membrane. 

In  the  protoplasm  is  a  varying  amount  of  rather  fine  granules, 
fat  droplets,  and  occasionally  vacuoles,  from  one  to  three  in  number. 
No  nucleus  is  seen.  Budding  forms  are  numerous.  The  bud  is 
a  projection  from  the  mother  cell  and  has  the  same  three  principal 
structural  elements,  each  being  continuous  with  the  corresponding 
parental  layer.  Both  capsule  and  clear  space  become  thinner  as 
they  pass  into  the  bud.  The  daughter  cell  reaches  approximately 
the  size  of  the  parent  before  it  separates  completely,  the  capsule 
gradually  becoming  heavier.  Isolation  of  the  cells  is  accomplished 
by  the  capsule  uniting  through  the  point  of  constriction  between 
the  cells.  No  vacuoles  are  seen  within  daughter  cells  so  long  as 
they  retain  connection  with  the  mother  cell. 

The  same  organism  is  found  in  teased  verrucous  tissue  mounted 
in  KOH  solution. 

After  two  fruitless  attempts  the  organism  was  obtained  on 
ordinary  media  inoculated  with  pus  from  the  small  abscesses  and 


Oidiomycosis  of  the  Skin  and  Its  Fungi  79 

with  teased  verrucous  tissue.  About  half  the  tubes  were  con- 
taminated with  a  few  colonies  of  a  white  staphylococcus. 

Agar-agar. — The  growths  on  plain-,  glycerin-,  and  glucose-agar 
are  identical  in  appearance  and  develop  more  slowly  on  the  first 
than  on  the  last  two.  Two  entirely  different  appearances  may 
result,  depending,  so  far  as  observed,  on  whether  the  culture  is 
kept  in  the  brood-oven  or  at  room  temperature. 

Cultures  in  the  brood-oven:  at  the  end  of  twenty-four  hours  a 
broad,  thin,  coarsely  granular,  whitish  growth  occurs  along  the 
stroke.  In  twenty-four  hours  more  it  doubles  in  proportions,  and 
the  granular  condition  develops  into  relatively  large,  prominent 
irregularities.  The  color  is  grayish- white  and  the  surface  moist. 
At  the  same  time  a  light,  hazy  down-growth  of  delicate  fibers 
penetrates  the  depth  of  the  medium.  The  heaping-up  on  the 
surface  progresses  rapidly.  In  the  center  the  growth,  instead  of 
being  purely  granular,  comes  to  resemble  a  heap  of  tangled  earth- 
worms, the  separate  coils  standing  out  distinctly  and  separated 
from  adjacent  coils  by  irregular  clefts.  As  one  passes  from  the 
lower  moist  surface  of  the  medium  to  the  upper,  somewhat  dried 
portion,  the  vermicular  appearance  gradually  passes  to  that  of 
the  coarsely  granular.  The  lateral  growth  is  made  up  of  delicate 
fibers  like  those  penetrating  the  medium  vertically.  It  does  not 
rise  above  the  level  of  the  medium,  but  is  intimately  associated 
with  it.  The  surface  presents  a  dull,  opaque,  frosted  white  appear- 
ance, finely  granular  and  marked  by  extremely  fine  rays,  originating 
in  the  central  portion.  By  reflected  light  the  peripheral  limitation 
seems  quite  abrupt,  but  by  transmitted  light  an  extension  beyond 
this  border  is  seen  in  the  shape  of  branching,  mossy,  tree-like 
rays  of  great  delicacy.  The  growth  into  the  depth  of  the  medium 
progresses  steadily  until  the  transparency  of  the  latter  is  destroyed. 

At  room  temperature:  the  growth  does  not  rise  above  the 
surface  of  the  medium  conspicuously,  but  becomes  intimately 
incorporated  with  it.  At  first  the  surface  appears  merely  wrinkled. 
The  folds  increase  in  size  rapidly,  and  there  are  corresponding 
depressions,  and  a  positive  consumption  of  the  medium  takes  place. 
The  surface  finally  resembles  a  piece  of  crumpled  cloth.  The 
medium  thus  impregnated  with  the  fungous  growth  may  be  broken 


8o  Contributions  to  Medical  Science 

into  soft,  brittle  fragments,  which,  when  smeared  over  the  surface 
of  a  fresh  tube,  give  a  uniform  growth  of  the  organism.  The  color 
is  grayish-white. 

Maltose  agar. — The  growth  here  rises  above  the  surface  in 
rounded  prominences,  and  the  centers  of  colonies  are  depressed. 
Threads  penetrate  the  medium  deeply.  The  surface  is  covered 
with  short  upright  spikes,  which  do  not  appear  like  true  aerial 
h}q)hae.  Microscopically,  they  are  composed  of  loosely  segmented 
threads,  the  segments  being  elongated  or  spherical.  Often  long 
segments  lie  closely  applied  side  by  side,  phalanx-hke.  Pink 
nucleus-like  bodies  are  seen  in  many.  In  addition,  numerous 
cells  are  more  or  less  filled  with  clear,  highly  refractive  globules 
(Fig.  i8a,  Plate  9).  There  are  in  addition  finer  non-segmented 
threads,  which  usually  contain  no  nucleus-like  structure.  The 
threads  penetrating  the  medium  have  a  similar  structure,  and 
give  off  a  few  conidia,  which  produce  buds. 

+70  Oxalic  acid  maltose  agar. — The  growth  is  less  pronounced 
than  on  other  agars,  and  is  less  intimately  incorporated  with  the 
medium.  The  microscopic  findings  are  the  same  as  on  maltose 
agar.  (Other  organisms  studied  do  not  grow  so  well  on  this 
medium.) 

The  reverse  surface  of  agar  slants  shows  no  pigmentation. 

Glycerin  agar  plate. — In  twenty-four  hours  minute  white  dots 
appear,  which  grow  to  a  millimeter  in  diameter  in  twenty-four 
hours  more.  The  naked-eye  appearance  is  that  of  dehcately 
chased,  almost  circular  crystals.  With  low  magnification,  the 
colonies  are  seen  to  consist  of  a  dense  central  area,  from  which 
threads  shoot  out  radially,  growing  less  numerous  as  the  periphery 
is  approached;  are  loosely  segmented,  and  produce  false,  loosely 
attached  branches,  coming  off  at  nearly  right  angles.  There  are 
no  lateral  or  terminal  conidia,  and  the  threads  are  slightly  tortuous. 
The  deep  colonies  show  the  same  structure  as  those  on  the  surface, 
and  are  of  equal  size.  There  is  no  aerial  growth.  The  appearances 
were  similar  on  glucose  agar  plates. 

Glucose  agar  stab. — A  finely  granular  development  occurs 
along  the  stab,  from  which  delicate  rays  extend  laterally  into 
the  medium.     Deeply  along  the  line  of  puncture  the  growth  is 


Oidiomycosis  of  the  Skin  and  Its  Fungi  8i 

less  pronounced  than  near  the  surface.  The  surface  of  the  medium 
is  soon  covered  with  a  thick  growth,  presenting  characteristic 
irregularities. 

Glucose  agar  anaerobic  stab. 


Gelatin  anaerobic  culture.      \ 

Bouillon  gelatin  stab  (aerobic). — Same  appearance  as  in  agar 
stab.  The  lateral  rays  are  seen  more  distinctly  on  account  of  the 
transparency  of  the  medium.  The  surface  growth  is  much  wrinkled, 
and  gradual  liquefaction  takes  place.  Plum-decoction  gelatin  is 
Hquefied  readily. 

Ox-blood  serum. — Development  proceeds  more  slowly  than  on 
the  agars.  The  surface  growth  is  not  so  vermicular,  though  this 
tendency  is  apparent.  The  lateral  portion  is  present  also,  but  the 
fine  structure  cannot  be  seen. 

Potato. — Development  is  rapid.  In  a  week's  time  the  whole 
surface  is  covered  with  a  light  fawn-colored  growth,  about  one- 
sixteenth  of  an  inch  deep.  The  surface  presents  a  densely  packed 
vermicular  structure.  At  the  upper  portion,  where  the  potato  has 
dried,  the  growth  is  powdery  white. 

Bouillon. — Identical  appearances  were  produced  on  plain  and 
on  a  2  per  cent  glucose  bouillon,  but  development  is  more  rapid  on 
the  glucose.  A  sediment  is  seen  at  the  end  of  twenty-four  hours. 
In  another  day  particles  appear  on  the  surface  and  adhering  to 
the  tube.  The  surface  growth  increases  until  it  is  a  soHd  floating 
layer,  which,  if  broken  up  by  shaking,  sinks  in  large  flakes.  If  the 
tube  is  undisturbed,  the  growth  at  the  bottom  also  becomes  dense, 
and  when  agitated  breaks  up  as  the  surface  growth  does.  The 
microscope  shows  a  great  deal  of  granular  material  and  broken-up 
cells,  besides  chains,  threads,  and  isolated  spherical  forms. 

The  top  growth  consists  of  densely  interwoven  mycelial  threads, 
which  bear  conidia.  There  are  a  few  spherical  budding  cells,  and 
a  gelatinous  and  granular  ground-substance. 

In  maltose  bouillon  the  mycelial  growth  predominated.  There 
were  a  few  spherical  budding  cells.  The  pink  nucleus-like  structure 
occurred  often. 

In  sugar-free  bouillon  the  threads  showed  little  tendency  to  be 
segmented. 


82  Contributions  to  Medical  Science 

From  agar  slants  which  grew  according  to  the  second  method, 
fragments  of  the  impregnated  medium  were  introduced  into 
glucose  bouillon  tubes.  Around  each  fragment  a  light  fluffy  tuft 
grew  to  considerable  dimensions.  Each  consisted  of  a  densely 
woven  mycelium.     Slowly  the  large  flakes  formed  as  described. 

Litmus  milk. — Growth  is  slower  than  in  bouillon.  A  large 
mass  appears  on  the  surface.  In  three  or  four  days  a  slight  reddish 
tint  appears  in  the  medium,  and  after  a  week  pronounced  acid 
reaction  is  noted. 

Hanging-drop  culture  in  glucose  bouillon . — Growth  occurs  readily 
at  room  temperature.  A  characteristic  feature  is  the  formation 
of  isolated  clumps  of  a  tortuous  mycelium,  with  almost  no  tend- 
ency to  shoot  out  into  the  surrounding  medium.  The  borders  are 
fringed  with  crooked,  tortuous  threads,  projecting  for  a  short 
distance,  the  segments  being  of  an  irregular  form.  The  impression 
is  that  of  a  tangled  mass  of  earth-worms,  the  microscopic  appear- 
ance here  suggesting  the  gross  appearance  on  solid  media.  Myce- 
lium originates  from  individual  cells  or  as  a  prolongation  from 
already  existing  threads,  and  is  of  a  branching  nature.  A  long, 
homogeneous,  translucent,  tapering,  encapsulated  thread  is  sent 
out  from  a  single  cell  or  chain  of  cells.  This  thread  may  be  suffi- 
ciently long  to  accommodate  from  ten  to  twenty  segments,  which 
may  form  successively,  or  one  or  two  well-formed  segments  may 
appear  at  the  middle  point  of  a  homogeneous  thread,  the  remaining 
portion  showing  no  differentiation  at  the  time  (Figs.  9  and  10, 
Plate  6).  Gradually  more  septa  are  formed.  The  cells  grow  or 
swell  so  that  constrictions  occur  at  the  point  of  division  and  the 
protoplasm  becomes  granular.  Eventually  the  segments  of  a 
thread  separate.  Many  are  dormant  and  when  watched  for  days 
show  no  change.  Others  start  new  threads.  Many  of  the  granules 
may  be  spoken  of  as  ''protoplasmic,"  and  some  seem  fatty;  but 
certain  others  appear  of  another  nature.  Cells  containing  these 
granules  increase  in  size  to  from  fifteen  to  twenty-five  microns, 
are  globular  or  oblong,  and  acquire  highly  refractive  globules, 
the  largest  of  which  are  a  fraction  of  a  micron  in  diameter.  These 
cells  disintegrate.     In  the  immediate  vicinity  there  develop  in  a 


Oidiomycosis  of  the  Skin  and  Its  Fungi  83 

few  days  large  numbers — from  fifty  to  a  hundred — of  young  cells. 
They  are  oblong,  lanceolate,  homogeneous,  and  soon  reach  a 
length  of  from  two  to  five  microns,  with  a  diameter  of  one-half 
of  that  extent.  They  gradually  become  separated,  increase  in 
size,  assume  adult  characteristics,  and  some  were  seen  to  sprout 
new  threads.  These  are  the  same  refractive  bodies  seen  in  mounts 
from  maltose  agar  tubes. 

In  a  hanging  drop  of  50  per  cent  iodide  of  potassium  solution, 
many  cells  become  filled  with  similar  bodies.  Cultures  from  the 
drop  were  sterile  after  two  weeks. 

The  organism  was  studied  in  a  hanging  drop  of  dog's  serum. 
Proliferation  occurred  solely  by  the  segmentation  and  breaking- 
up  of  mycelium  into  elongated,  oval,  or  spherical  cells,  which 
elongated  to  form  new  threads.  Growth  was  very  rapid.  The 
nucleus-like  structure  and  many  highly  refractive  spherules  ap- 
peared in  numerous  cells. 

Fermentation  tests. — Glucose  solutions  are  fermented  readily; 
lactose,  maltose,  and  saccharose  solutions  are  not  affected.  Alco- 
hol, carbon  dioxide,  and  acetic  acid  result  in  the  process  of  fermen- 
tation. 

Potassium  iodide  cultures. — Vigorous  development  occurs  in 
one  and  five  per  cent  solutions  in  bouillon,  with  no  alterations  in 
the  morphology  of  the  organism.  A  considerable  amount  of 
brownish-black  pigment  forms  in  the  bottom  of  the  tube. 

Indol. — None  is  produced  in  the  Theobald  Smith  sugar-free 
bouillon. 

HiSTOPATHOLOGY.— On  two  separate  occasions,  under  freezing 
anesthesia,  verrucous  tissue,  containing  miliary  abscesses,  was 
removed,  fixed  in  bichloride  of  mercury,  and  imbedded  in  paraffin. 
The  two  specimens  show  the  same  changes. 

Horny  layer. — Absent  in  small  foci,  exposing  the  rete.  In  other 
places  it  is  greatly  heaped  up,  forming  plugs  which  fit  into  depres- 
sions of  the  rete  and  rise  above  the  surface  level.  Composed 
largely  of  flattened  nucleated  cells,  in  the  midst  of  which  are 
polymorphonuclear  leukocytes,  bacteria,  and  debris. 

The  stratum  lucidum  is  not  observed  in  the  diseased  area. 


84  Contributions  to  Medical  Science 

Granular  layer. — Present  except  where  desquamation  is  extreme. 
Where  the  rete  is  thick  the  granular  layer  acquires  proportionate 
thickness. 

Rete. — The  enormous  proHferation  and  down-dipping  charac- 
teristic of  the  diseases  are  conspicuous.  Conditions  of  the  rete 
cells  differ  Uttle  from  those  in  the  preceding  case.  Edema  is  more 
marked.  Individual  cells  are  large,  their  nuclei  stain  well,  the 
prickles  are  distinct  and  stain  faintly  with  protoplasmic  dyes. 
There  are  isolated  examples  of  epithelial  cells  including  polymor- 
phonuclear leukocytes.  These  latter  cells  are  not  so  uniformly 
and  densely  distributed  as  in  the  first  case.  No  plasma-  or  mast- 
cells  are  found  in  the  rete. 

The  intra-epithelial  abscesses  are  larger  than  those  of  the 
first  case,  reaching  at  times  the  diameter  of  a  milhmeter,  and 
they  frequently  connect  with  abscesses  on  the  cutis.  They 
contain  polymorphonuclear  leukocytes,  epithelial  cells,  granular 
detritus,  and  the  organism  peculiar  to  the  case.  From  two  to  four 
giant  cells  may  be  found  in  the  section  of  an  abscess,  commonly 
near  the  periphery.  The  nuclei  are  arranged  peripherally.  Free 
epithelial  cells  are  found  less  frequently  than  in  the  previous  case, 
and  they  do  not  present  so  many  examples  of  phagocytosis  or  cell 
invasion.  They  degenerate  much  more  readily,  and  usually  are 
seen  as  acidophilic  granular  masses.  From  one  to  several  organ- 
isms may  be  seen  in  a  single  section  of  an  abscess;  on  the  other 
hand  several  sections  may  be  examined  without  encountering  any 
organism. 

Cutis. — Normal  structures  are  obhterated  by  a  mass  of  infiltrat- 
ing cells,  among  which  polymorphonuclear  leukocytes  and  plasma 
cells  predominate,  the  latter  being  present  in  smaller  numbers  than 
in  the  first  case.  They  have  the  same  relation  to  the  polymor- 
phonuclears and  to  new-forming  connective  tissue.  Hyalin  bodies 
are  met  with  less  frequently.  The  polymorphonuclears  form 
distinct  subcutaneous  abscesses,  and  they  are  also  diffusely  dis- 
tributed in  smaller  numbers.  In  the  abscesses  are,  besides  the 
leukocytes,  portions  of  connective-tissue  fibers,  an  occasional 
elastic  fiber  fragment,  proliferating  connective-tissue  cells,  granular 


Oidiomycosis  of  the  Skin  and  Its  Fungi  85 

material,  giant  cells,  and  the  organism.  Mast-  and  plasma-cells 
are  found  rarely  in  the  periphery  of  the  abscesses. 

Mast-cells  of  the  types  already  mentioned,  and  in  like  dis- 
tribution, are  numerous. 

Giant  cells  of  the  tuberculous  type  are  relatively  numerous,  and 
are  found  not  only  in  the  abscesses,  but  also  in  connective  tissue 
less  markedly  infiltrated.  None  are  found  containing  the  organism. 
The  tubercle  bacillus  could  not  be  found  in  properly  stained  sections. 

The  organism. — It  is  found  both  in  the  epithelium  and  cutis. 
In  the  former  it  is  surrounded  by  varying  numbers  of  leukocytes, 
which  often  constitute  small  abscesses.  In  the  cutis  it  is  found 
both  in  distinct  abscesses  and  where  there  are  fewer  inflammatory 
cells.  The  characters  are  the  same,  whether  found  in  the  epithe- 
lial layer  or  the  cutis.  The  typical  form  is  spherical.  Occasionally 
an  elongated  cylindrical  form  is  seen,  either  alone  or  with  a  bud  or 
large  spherical  form  attached  to  one  end.  The  average  diameter  is 
twelve  microns.  Budding  as  described  by  previous  writers  is 
common.  A  limiting,  homogeneous,  translucent,  doubly  con- 
toured capsule,  a  clear  space  internal  to  this,  inconstantly  seen, 
and  a  central  protoplasm  are  the  principal  structural  details. 
In  methylene  blue  preparations  the  capsule  usually  stains  deeply, 
and  often  its  outer  surface  is  irregular,  giving  the  impression  of 
adhering  foreign  matter.  The  protoplasm  is  granular,  vacuoles 
occurring  in  occasional  cells.  In  a  pair,  the  protoplasm  of  one 
cell  may  stain  deeply  with  methylene  blue,  and  that  of  the  other 
not  at  all.  Other  nuclear  stains  are  treated  with  like  indifference. 
With  all  stains  one  finds  inconstantly  in  the  periphery  of  the 
central  protoplasm  a  number  of  minute  spherical  bodies.  Bodies 
similar  to  these  are  frequently  seen  free,  but  their  identity  is  uncer- 
tain. Old  cells  in  which  there  is  no  clearly  recognizable  central 
protoplasm  do  not  stain.  With  acid  orcein,  the  capsule  and  a 
limiting  protoplasmic  membrane  stain  deeply.  The  protoplasm 
has  but  slight  affinity  for  hematoxylin.  Fuchsin  is  taken  up 
moderately,  but  not  in  a  distinctive  manner.  With  carbol-toluidin 
blue  the  same  results  are  obtained  as  with  methylene  blue.  No 
constant  nor  distinctive  staining  affinities  have  been  found. 


86  Contributions  to  Medical  Science 

Animal  Experiments. —  Guinea-pigs.  i.  Intraperitoneal  inoculation  of  teased 
verrucous  tissue  in  normal  salt  solution.  The  animal  died  after  two  days,  of  staphylo- 
coccus septicemia. 

2.  Intraperitoneal  inoculation  of  twenty  minims  of  glucose  bouillon  culture 
grown  in  the  incubator  for  one  week.     The  inoculation  was  without  apparent  results. 

3.  Twenty  minims  of  the  culture  used  in  guinea-pig  2  were  inoculated  beneath 
the  abdominal  skin.  An  abscess  developed  after  five  days.  It  ruptured  sponta- 
neously. The  organism  was  found  in  the  pus  mounted  in  KOH  solution,  and  cultures 
on  agar  yielded  the  fungus  inoculated. 

4.  Four  centimeters  of  a  ten-day-old  glucose  bouillon  culture  inoculated  beneath 
the  skin  of  the  back.  The  animal  ran  a  temperature  for  three  days,  appeared  sick, 
and  refused  food.  However,  abscess  formation  did  not  occur,  and  complete  recovery 
took  place. 

5.  Subcutaneous  inoculation  of  two  centimeters  of  a  sugar-free  bouillon  culture. 
Four  days  later  was  found  dead.  The  autopsy  showed  pulmonary  tuberculosis. 
Microscopic  examination  corroborated  the  gross  findings.     Cultures  were  sterile. 

Mice.  I.  Twelve  minims  of  a  glucose  bouillon  culture  injected  subcutaneously. 
Recovery  apparently  complete. 

2.  Two  centimeters  of  a  glucose  bouillon  culture  inoculated  subcutaneously. 
Died  after  two  days.  Cultures  yielded  pus  cocci.  The  organism  was  not  discovered 
in  sections. 

Dogs.  I.  November  28,  1900.  Three  centimeters  of  a  five-day-old  glucose 
bouillon  culture  inoculated  intravenously.  Killed  one  month  later.  Cultures  were 
sterile.  No  gross  pathological  conditions  except  a  few  small  subcapsular,  scar-like 
lesions  of  the  kidneys.  Under  the  microscope  these  were  miliary  nodules,  consisting 
of  new  connective-tissue-  and  plasma-cells.  No  fungus  cells  recognizable.  No  tubercle 
bacilli  present.     Other  organs  were  normal. 

2.  December  3,  1900.  Four  centimeters  of  a  five-day-old  glucose  bouillon 
culture  injected  subcutaneously.     No  effect.     Animal  living  (March  2,  1901). 

Rabbits,  i.  February  13,  1901.  Died  five  minutes  after  an  intravenous  inocula- 
tion.    Pulmonary  embolism. 

2.  February  14,  1 90 1.  Two  centimeters  of  bouillon  suspension  of  a  culture  on 
-|-io  oxalic  acid  maltose  agar,  intravenously.  Died  two  days  later.  Enlarged,  pale 
kidneys.  Cultures  sterile.  In  the  lungs  were  a  few  small  foci  of  epithelial  prolifera- 
tion, and  infiltration  with  lymphocytes  and  polymorphonuclear  leukocytes.  The 
liver  and  kidneys  were  fatty.     Other  organs  normal. 

White  rats.  i.  November  11,  1900.  Intraperitoneal  inoculation  of  one  and 
one-half  cubic  centimeters  of  a  four-day-old  glucose  bouillon  culture.  No  symptoms 
followed.     The  animal  is  still  living  (March  3,  1901). 

2.  February  14,  1901.  Subcutaneous  inoculation  of  two  centimeters  of  sugar- 
free  bouillon  culture.     No  effect.     Animal  living  (March  3,  1901). 

Summary  of  Biologic  and  Pathogenic  Properties. — Young 
colonies  are  grayish,  round,  elevated,  granular,  and  early  become 
incorporated  with  agar  and  blood-serum,  and  gelatin  media. 
Colonies  fuse  readily,  and  eventually  form  an  elevated,  deeply 
and  coarsely  wrinkled  surface,  of  a  dirty  gray  color.     The  growth 


Oidiomycosis  of  the  Skin  and  Its  Fungi  87 

is  teased  apart  with  difficulty.  The  posterior  surface  of  the  slant 
does  not  have  the  rich  golden-brown  color  often  seen  in  cultures 
of  fungi.  In  gelatin  stabs  lateral  rays  are  formed;  no  liquefaction. 
In  liquid  media  a  membranous  top  growth  forms  and  coherent 
flakes  and  tufts  develop  as  a  sediment  and  adhering  to  the  tube 
surface.     Milk  is  coagulated  and  acidified;   no  indol-formation. 

Mycelium  always  predominates,  but  it  is  accompanied  by  a 
varying  amount  of  budding  on  the  part  of  spherical  or  oval  cells. 
No  demonstrable  nucleus.  The  formation  of  endospores  in  one 
observation  seemed  fairly  positive;  but  further  study  of  this 
change  in  the  protoplasm  indicates  rather  a  plasmolysis,  which 
does  not  necessarily  involve  the  death  of  the  cell.  A  study  of 
Figs.  i8a  and  iSb,  Plate  9,  shows  the  method  of  development 
of  these  bodies.  The  objections  to  their  being  considered  as 
ascospores  are  (i)  the  large  number  which  may  be  found;  and 
(2)  they  have  never  been  observed  to  grow  into  spherical  cap- 
sulated  cells  or  fibers. 

The  organism  reproduces,  then,  by: 

1.  A  segmented  branching  mycelium. 

2.  The  division  and  separation  of  mycelial  segments,  producing 
spherical  cells  or  "spores." 

3.  Lateral  conidia  (for  a  time). 

4.  Budding  of  spherical  and  oval  cells. 

5.  Endogenous  spore-formation  (?). 

Subcutaneous  abscesses  may  form  in  guinea-pigs  and  rabbits. 
Dogs  and  mice  are  not  susceptible.  Its  pathogenic  effect  on  animals 
is  weak.     An  inoculated  scarification  in  man  healed  promptly. 

Case  IV. — Clinical  history. — Mr.  F.  M.,  a  resident  of  Chicago, 
presented  himself  at  the  clinic  December  23,  1899.  He  was 
forty-seven  years  of  age,  single,  with  good  general  history. 

His  present  disorder  dated  from  November,  1897,  when  the 
skin  of  the  right  forearm,  three  inches  below  the  elbow-joint,  was 
scraped  when  in  contact  with  a  wooden  truck.  The  resulting 
wound  covered  itself  with  a  crust,  which  later  fell  and  was  suc- 
ceeded by  another  and  yet  another,  no  healing  resulting. 

The  patch,  when  examined,  was  found  to  be  in  the  form  of  a 


88  Contributions  to  Medical  Science 

distinctly  defined  band,  about  six  centimeters  in  greatest  width, 
completely  encircling  the  arm  from  in  front  backward  and  meeting 
posteriorly  by  a  narrow  line  of  contact  between  the  two  rounded 
borders  of  the  fillet,  as  if  the  process  had  advanced  from  the  anterior 
to  the  posterior  surface.  The  affected  area  was  of  a  dull,  reddish 
hue,  moist  throughout,  secreting  a  sero-purulent  fluid,  granulating, 
and  the  source  of  a  disgusting  fetor.  Its  surface  was  irregular,  and 
there  were  islets  of  pinkish  hue  between  the  elevations  and  depres- 
sions of  the  florid  granulations  visible.  Here  and  there  thin  patches 
of  false  membrane  had  formed. 

At  the  present  date  the  arm  of  the  patient  is  better,  and  practi- 
cally healed  after  treatment  with  iodide  of  potassium. 

Examination  of  the  Fresh  Tissue  and  Pus. — On  December 
23,  1899,  large  villiform  processes  were  teased  and  examined  in 
30  per  cent  KOH  solution.  The  patient  had  received  no  potassium 
iodide.  The  specimen  was  kept  under  observation  for  three 
days,  at  the  end  of  which  time  the  forms  to  be  described  were 
relatively  intact. 

Bodies  possessing  the  characteristics  ascribed  to  blastomycetes 
were  abundant.  The  typical  solitary  form  was  spherical,  and  had 
a  doubly  contoured,  structureless,  clear,  highly  refractive  capsule, 
and  a  central  protoplasm  which  was  finely  or  coarsely  granular, 
and  sharply  differentiated  from  the  capsule.  Often  certain  clear, 
structureless,  highly  refractive  bodies  were  suspended  in  the 
protoplasm.  These  were  considered  vacuoles.  The  number  in  a 
single  ceU  varied  from  one  to  several,  but  they  were  often  absent; 
fat  globules  common.  Certain  other  granules  of  nearly  constant 
size  were  observed  frequently,  usually  arranged  around  the  periph- 
ery of  the  cell.  They  may  have  been  spores.  Nothing  was 
seen  of  a  distinct  nucleus.  One  chain  of  four  organisms  was 
observed,  each  united  to  its  neighbor  by  a  portion  of  capsular 
substance,  the  protoplasm  of  each  being  enclosed  in  its  own  cap- 
sule. Organisms  with  two  buds  were  seen.  A  sort  of  abortive 
budding  occurred  occasionaUy,  the  large,  cylinder-like  body 
having  a  slight  mesial  constriction.  Some  of  the  forms  exhibited 
a  cup-Uke  depression  of  the  capsule,  indenting  the  protoplasm. 
A   "clear  zone"   was   ijot   always   demonstrable.     Many  of   the 


Oidiomycosis  of  the  Skin  and  Its  Fungi  89 

organisms  were  degenerating,  even  in  perfectly  fresh  preparations. 
At  first  the  capsule  becomes  marked  with  meridian-like  lines, 
which  increase  in  number  and  prominence,  and  are  seen  to  involve 
the  whole  thickness  of  the  capsule,  eventually  appearing  as  lines 
of  fracture.  The  fragments  finally  become  separated,  forming 
a  layer  of  debris  around  the  protoplasm.  The  latter  substance 
in  the  beginning  of  the  process  acquires  coarse  granules,  which 
merge,  and  as  this  takes  place  the  volume  of  the  mass  is  contracted. 
The  protoplasm  finally  is  converted  into  a  perfectly  clear  sub- 
stance.    The  final  dissolution  stage  could  not  be  found. 

The  diameter  of  the  solitary  adult  form  averaged  from  twelve 
to  fourteen  microns;  none  exceeded  sixteen. 

Pus  taken  from  small  abscesses  showed  organisms  of  the  same 
character. 

On  January  3,  18,  and  February  10,  further  examinations  of  the 
pus  and  fresh  tissue  in  potassium  hydrate  solutions  were  made. 
The  findings  recorded  were  duplicated,  and,  moreover,  what  is 
considered  as  a  small  form  of  the  organism  was  seen.  It  may  be 
that  this  was  an  involution  form  induced  by  the  potassium  iodide 
administered.  The  identity  of  this  form  rests  on  a  purely  mor- 
phological basis,  because  of  the  failure  to  obtain  cultures.  They 
measured  from  four  to  six  microns  in  diameter,  had  delicate  but 
distinct  capsules,  multiplied  by  budding,  and  were  prone  to  form 
short  threads  of  four  to  six  or  eight  segments.  The  protoplasm 
was  finely  granular  and  showed  no  vacuoles  or  nucleus.  Forms 
intermediate  between  this  and  the  larger  were  not  seen.  This 
small  form  was  found  only  in  teased  preparations  of  the  tissue, 
not  in  pus  from  miliary  abscesses. 

On  February  27  were  found,  besides  the  typical  adult  form, 
a  few  large  individuals  of  twenty  to  thirty  microns  in  diameter. 
The  capsule  was  only  sUghtly  thicker  than  that  of  the  average 
form.  The  center  was  made  up  of  granular  protoplasm,  in  which 
were  suspended  bodies  of  varying  degrees  of  refraction,  measuring 
from  one  to  four  microns  in  diameter.  They  show  no  definite 
structure. 

A  further  examination  was  made  on  May  2,  1900,  when  he  had 
been    under   potassium    iodide    for    several    months.     Extensive 


90  Contributions  to  Medical  Science 

healing  had  taken  place,  leavmg  a  thick,  smooth  scar,  which 
still  contained  a  few  mihary  abscesses.  These  yielded  the  organism 
in  its  ordinary  spherical  form,  exhibiting  no  marked  degeneration, 
as  one  might  expect  to  find  in  such  a  rapid  subsidence  of  the  process. 
However,  they  were  much  fewer. 

Cultures. — Culture  tubes  of  the  ordinary  media  were  inocu- 
lated many  times,  both  from  the  teased  verrucous  tissue  and  from 
the  pus  of  the  miliary  abscesses.  Numerous  ordinary  bacteria 
grew  on  the  tubes,  but  an  organism  resembling  the  usual  "blas- 
tomyces"  or  the  form  seen  in  the  tissues  never  was  obtained. 
Control  examinations  were  always  made  at  the  same  time  and 
from  the  same  material  used  in  inoculations.  It  was  found  invari- 
ably that  the  pus  or  the  teased  tissue  experimented  with  contained 
numerous  organisms.  Each  time  inoculations  were  made,  how- 
ever, there  grew  on  one  or  two  tubes  one  or  more  colonies  of  a 
thread-like  fungus,  which  penetrated  the  medium,  the  center  of 
the  colony  showing  a  small  conical  elevation,  but  producing  no 
true  surface  growth.  Film  preparations  showed  threads  with 
small  lateral  pedunculated  conidia.  Transplantation  on  other 
media  produced  no  growth,  and  further  study  could  not  be  made. 
Maltose-agar  was  not  used  at  that  time.  The  early  appearance 
of  this  fungus  was  exactly  like  that  in  a  more  recent  case  in  the 
light  of  which  it  seems  probable  that  we  were  dealing  with  the 
specific  organism. 

Animal  Experiments. —  Fresh  tissue  teased  in  bouillon  was  used. 
Guinea-pigs.    i.    Intra-abdominal    inoculation.     Death    in    three    days.     Peri- 
tonitis.    Microscopically,  mild  fatty  degeneration  of  the  liver  and  kidneys. 

2.  Subcutaneous  inoculation  and  scarification.  The  latter  healed  promptly; 
the  former  was  swollen  for  a  few  days,  but  healed  without  suppuration.  Death  a 
month  later,  preceded  by  emaciation.  Postmortem  findings  normal.  Macro- 
scopically,  cloudy  swelling  of  the  organs. 

3.  Subcutaneous  inoculation.  Induration  slight,  which  disappeared  slowly. 
Death  three  weeks  later.  There  were  no  gross  findings.  Microscopically,  only 
cloudy  swelling  of  liver  and  kidneys. 

Owing  to  the  absence  of  special  tissue  changes,  and  to  the  fact  that  several 
other  animals  died  about  this  time  from  obscure  causes,  it  seems  improbable  that  the 
inoculations  were  responsible  for  the  death  of  the  last  two  guinea-pigs. 

Histopathology. — Four  specimens  were  removed  at  different 
dates,  representing  various  conditions. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  91 

The  low-power  view  is  that  of  a  promiscuous  mixture  of  epithe- 
lium and  altered  papillary  tissue.  The  latter  is  in  the  form  of 
tortuous  processes  and  islands,  always  sheathed  by  epithelium. 

The  horny  layer  is  excessive.  It  follows  the  irregular  pro- 
jections and  depressions  of  the  papillae,  and  furthermore  in  the 
depressions  forms  loose  masses  and  whorls  of  flattened  cells,  many 
of  which  are  nucleated.  Among  them  are  occasional  polymor- 
phonuclear leukocytes  and  a  few  red  blood  corpuscles.  Large 
masses  and  layers  of  cocci,  which  stain  by  Gram's  method,  are 
present.  Acid  fuchsin  shows  many  variously  sized  hyalin  bodies 
both  within  and  between  the  cells. 

Rete.— It  follows  the  meanderings  of  the  papillary  tissue  as 
a  thick  sheath.  Its  cells  are  large,  vesicular,  their  nuclei  stain 
well,  and  the  prickles  are  conspicuous  and  often  widely  separated. 
There  is  no  stratum  lucidum,  and  only  traces  of  a  granular  layer 
are  found.  Whorl  formation,  with  partial  comification,  is  seen 
often  in  the  depth  of  the  rete.  These  whorls  contain  leukocytes 
in  the  center  and  between  the  cells,  but  none  have  been  found 
containing  organisms.  Mitoses  are  common.  The  basal  layer 
retains  distinct  columnar  formation,  but  it  is  often  subject  to 
distortions.  Large  circular  spaces  between  epithelial  cells  speak 
for  a  marked  edema  of  the  rete.  The  number  of  infiltrating  leu- 
kocytes in  the  rete  is  small  compared  with  other  cases.  The 
polymorphonuclear  cells  predominate,  but  mononuclears  and 
eosinophiles  are  also  seen,  the  latter  in  conspicuous  numbers. 
Red  blood  cells  are  relatively  numerous  between  the  rete  cells. 
The  leukocytes  are  gathered  into  small  masses  in  many  places  to 
form  intra-epithelial  abscesses,  which  are  smaller  than  in  the 
second  case,  but  usually  larger  than  those  in  the  first.  They 
contain,  besides  the  leukocytes,  loosened  epithelial  cells  of  types 
already  mentioned,  occasional  eosinophiles  and  red  blood  cells, 
the  organisms,  and  the  usual  granular  material.  No  plasma- 
or  mast-cells  are  seen  within  the  abscesses  or  free  in  the  rete. 
Often  there  is  a  group  of  organisms  with  only  a  few  surrounding 
leukocytes.  The  conditions  of  the  epithelium  within  and  surround- 
ing the  abscesses  are  like  those  described  in  connection  with  the 
two  preceding  cases. 


92  Contributions  to  Medical  Science 

Cutis. — In  the  active  verrucous  condition,  the  epithelium 
penetrates  the  cutis  promiscuously,  but  there  are  no  isolated  masses 
or  whorls.  No  papillary  layer  as  such  is  to  be  distinguished. 
The  blood-vessels  are  widely  dilated,  containing,  besides  the  red 
and  polymorphonuclear  cells,  a  relatively  large  number  of  eosino- 
philes.  No  plasma  cells  are  seen  in  the  vessels.  In  the  superficial 
cutis  are  many  small  hemorrhages.  These  are  so  numerous  that 
in  a  good  eosin  stain  the  field  appears  quite  red.  The  distribution 
of  infiltrating  cells  and  of  particular  varieties  is  irregular.  Polymor- 
phonuclear leukocytes,  lymphocytes,  plasma-  and  mast-cells, 
eosinophiles,  young  connective-tissue  and  giant  cells,  red  blood 
cells,  and  the  organism  constitute  the  mass  of  cells. 

The  polymorphonuclear  leukocytes  here,  as  in  other  cases, 
collect  in  masses  to  form  small  subcutaneous  abscesses,  and  are 
moreover  distributed  diffusely  in  small  numbers.  Plasma  cells 
are  present  in  characteristic  masses  and  colimms,  but  in  much  less 
numbers  than  in  the  preceding  cases.  In  the  verrucous  tissue  they 
exist  only  in  small  collections.  Where  scar  tissue  is  forming 
actively,  they  are  distributed  in  accordance  with  the  amount  of 
scar  tissue  present.  Where  the  latter  is  marked,  plasma  cells 
are  less  numerous  than  in  places  where  such  tissue  is  beginning  to 
form.  This  is  in  harmony  with  the  conception  that  plasma  cells 
are  sacrificed  in  the  formation  of  inflammatory  connective  tissue. 
Examples  of  cells  which  have  lost  part  or  nearly  all  of  their  granular 
protoplasm  are  common.  It  is  noted  also  in  this  tissue  that  the 
last  plasma  cells  to  leave  the  field  are  those  situated  around  the 
blood-vessels.  Hyalin  degeneration  of  the  protoplasm  occurs, 
but  is  a  less  conspicuous  feature  than  in  the  first  case. 

Mast-cells  in  small  numbers  are  diffusely  distributed,  being 
absent,  however,  in  dense  collections  of  polymorphonuclear  leu- 
kocytes. 

In  the  active  verrucous  process  eosinophiles  are  common,  occur- 
ring singly  and  in  small  groups.  Their  granules  stain  equally  well 
with  eosin  or  acid  fuchsin. 

Giant  cells  have  a  promiscuous  distribution,  being  found  in 
mihary  abscesses,  in  neighboring  acutely  involved  tissue,  and  to 
a  less  degree  in  the  more  deeply  lying  scar  tissue,  where  ordinary 


Oidiomycosis  of  the  Skin  and  Its  Fungi  93 

infiltration  is  not  marked.  They  are  of  the  tuberculous  type,  and 
commonly  contain,  besides  the  intrinsic  nuclei,  polymorphonuclear 
and  mononuclear  leukocytes,  and  some  larger  (connective-tissue  ?) 
cells.  None  contained  the  fungus,  so  far  as  observed.  Tubercle 
bacilli  could  not  be  demonstrated  by  the  usual  staining  methods. 

Abscesses.— -Those  seen  in  the  subcutaneous  tissue  may  be  as 
large  as  a  millimeter  in  diameter.  The  intra-epithelial  are  usually 
smaller.  Giant  cells  have  not  been  found  in  the  latter,  but  degener- 
ating epithelial  cells,  phagocytosis,  and  cell  invasion  are  conspicu- 
ous. The  including  cells  are  epithelial  usually,  and  the  included 
bodies  are  leukocytes  and  small  globules,  considered  as  free  and 
altered  nuclear  material.  Besides  the  above  cells,  only  polymor- 
phonuclears, and  in  small  abscesses  an  occasional  eosinophile,  are 
found. 

The  subcutaneous  abscesses  are  made  up  of  polymorphonuclear 
and  mononuclear  leukocytes,  connective-tissue  and  giant  cells, 
portions  of  adult  fibrous  tissue,  fragments  of  elastic  tissue,  occa- 
sional mast-  and  plasma-cells,  and  the  organism  pecuKar  to  the 
case.     The  polymorphonuclear  leukocyte  is  the  prevailing  cell. 

The  organism  is  found  in  the  subcutaneous  and  intra-epithelial 
abscesses  and  frequently  in  connective  tissue  without  true  abscess 
formation.  It  is  always  surrounded  by  infiltrating  cells,  often  by 
a  very  small  number.  It  occurs  characteristically  in  clusters 
of  from  two  or  three  to  six  or  eight.  Single  forms  are  rare.  Iso- 
lated pairs  are  seen  occasionally.  In  a  group  the  sizes  of  the  indi- 
viduals vary,  the  adults  or  larger  forms  measuring  about  fourteen 
microns  in  diameter.  Budding  forms  are  numerous  and  may 
be  seen  in  all  stages.  An  adult  cell  may  send  off  two  or  three  buds, 
which  are  of  different  sizes,  indicating  separate  development  of 
the  different  buds.  Individual  organisms  have  the  structure 
typical  of  this  variety  of  fungus,  i.e.,  capsule,  clear  space,  and 
central  protoplasm.  The  capsule  has  a  peculiar  substance  depos- 
ited on  its  surface,  which  gives  it  a  rough,  somewhat  ragged, 
contour.  This  may  be  a  secretion  of  the  organism  or  a  reactionary 
deposit  from  the  tissue  fluids.  It  appears,  however,  to  be  an 
integral  part  of  the  capsule,  and  in  a  group  of  organisms  seemingly 
acts  as  an  agglutinating  substance.     It  stains  deeply  with  eosin 


94  Contributions  to  Medical  Science 

in  Zenker-hardened  specimens,  and  a  bluish-green  with  polychrome 
methylene  blue.  There  is  nothing  pecuhar  about  the  clear  space. 
It  is  usually  present  in  the  hardened  tissue.  In  the  central  proto- 
plasm of  young  cells  no  structure  can  be  made  out.  As  they  grow 
larger,  however,  a  granular  condition  is  apparent.  In  Zenker 
specimens  stained  deeply  with  eosin  a  spongy  structure  is  brought 
out  well.  Neutral  orcein  also  stains  this  substance.  Moreover, 
in  the  older  cells  it  is  usual  to  see  from  ten  to  twenty  or  thirty 
minute  spherical  bodies  arranged  peripherally  in  the  protoplasmic 
substance,  having  a  diameter  of  one-half  to  one  micron.  They 
stain  well  with  hematoxyUn  and  with  methylene  blue.  A  ruptured 
cell  was  found,  in  which  these  bodies  were  lying  partly  outside 
of  the  cell.  One  may  suppose  that  they  are  endospores.  Vacuoles 
are  often  seen  in  the  larger  cells.  They  are  structureless  and  their 
definition  smooth  and  sharp.  The  organism  has  no  demonstrable 
nucleus.  Separating  the  protoplasm  from  the  clear  space  is  a 
well-defined  membrane. 

The  efect  of  potassium  iodide  on  the  lesion. — Gradually  the 
verrucous  condition  subsided,  and  a  smooth,  ghstening,  scaling, 
thick,  leathery,  red  scar  resulted.  Many  minute  abscesses  were 
visible  just  beneath  the  epithehum.  A  portion  of  this  tissue  was 
removed  for  examination,  fixed  in  absolute  alcohol,  and  imbedded 
in  parafl&n.  It  cut  with  uniform  fibrous  resistance,  the  cut  surface 
being  pale  and  relatively  bloodless.  The  cicatricial  portion  was 
one  centimeter  deep.  It  showed  a  thickened  homy  layer  and 
stratima  granulosum.  The  rete  is  moderately  thickened,  but 
presents  no  positive  down-growths,  the  deeper  surface  being  com- 
paratively smooth.  Correspondingly,  the  normal  rete  pegs  are 
lacking,  and  the  papillary  layer  is,  of  course,  absent.  The  sub- 
epithelial tissue  consists  almost  entirely  of  young  connective- 
tissue  fibers  arranged  in  planes  parallel  to  the  surface.  There  are 
occasional  groups  of  plasma  cells  in  close  proximity  to  blood  cells. 
Mast-cells  are  numerous,  whereas  they  were  scarce  in  the  verrucous 
condition.  They  are  distributed  with  no  reference  to  any  special 
structure  or  condition.  They  are  distinctly  of  the  connective- 
tissue  cell  type.  There  are  no  eosinophiles.  Only  traces  of 
elastic   tissue   are   present.     While   intra-epithelial   abscesses   are 


Oidiomycosis  of  the  Skin  and  Its  Fungi  95 

not  present,  several  abscesses  are  encountered  in  the  subcutaneous 
tissue.  They  contain  a  few  organisms,  which  often  are  in  a  state 
of  disintegration. 

Case  F.* — Chnical  history. — C.  W.  T.,  aged  thirty-three, 
carpenter  by  occupation.  Had  always  been  unusually  strong  and 
vigorous.  Seven  years  previous  to  his  coming  to  the  hospital  he 
noticed  a  pimple  on  the  right  shoulder,  which  became  covered 
with  a  crust  which  gradually  increased  in  size. 

The  man's  general  health  had  been  unaffected,  the  disorder 
causing  him  no  pain,  though  at  times  it  was  sensitive  to  the  rubbing 
of  his  clothing  or  other  irritation.  Attempts  at  cultures  and  animal 
inoculations  both  with  pus  and  fresh  tissue  gave  no  positive  results 
beyond  cultures  of  pus  organisms.  The  large  number  of  giant 
cells  found  in  the  corium  led  to  a  diagnosis  of  probable  cutaneous 
tuberculosis,  with  a  possible  epitheliomatous  compUcation,  as  there 
was  marked  hyperplasia  of  the  rete. 

He  was  again  in  the  Cook  County  Hospital,  under  the  care  of 
the  late  Dr.  D.  D.  Bishop,  from  March  8  to  April  19,  1895.  Dur- 
ing this  time  the  cutaneous  disorder  continued  to  progress,  and  the 
man  gradually  lost  strength  and  became  decidedly  emaciated. 
He  died  soon  after,  of  what  his  physician  called  acute  miliary 
tuberculosis,  the  postmortem  showing  mihary  tubercles  in  the 
lungs  and  in  other  internal  organs.  It  was  reported  that  in  these 
lesions  tubercle  bacilli  were  discovered,  and  that  guinea-pigs 
inoculated  with  tissue  developed  generalized  tuberculosis. 

During  the  eight  months  in  which  he  was  under  observation, 
numerous  attempts  were  made  to  obtain  cultures,  but  nothing  was 
ever  found  except  pus  organisms.  A  number  of  guinea-pigs  were 
inoculated  at  different  times  with  bits  of  tissue  both  from  older 
and  from  recent  lesions.  The  majority  of  animal  inoculations 
gave  entirely  negative  results,  no  evidence  of  tuberculosis  being 
obtained  in  any  of  them.  Two  guinea-pigs  died  of  septic  infection 
a  few  days  after  inoculation.     - 

Tissue  from  the  older  lesions  showed  a  markedly  infiltrated 
cutis,  with  many  giant  cells,  some  of  which  were  unusually  large. 

■  The  following  report  is,  in  brief,  as  given  by  Professor  Montgomery,  who  discovered  the  nature  of 
the  case. 


96  Contributions  to  Medical  Science 

In  some  places  as  many  as  forty  giant  cells  could  be  distinguished 
in  a  single  field  under  the  microscope.  Hyperplasia  of  the  rete 
was  very  pronounced,  the  process  extending  deep  into  the  cutis, 
with  irregular  shape  and  branches.  In  these  epitheliomatous 
processes  were  miliary  abscesses  of  various  sizes,  some  of  them 
bounded  by  markedly  flattened  and  shghtly  hornified  epithelium. 
In  other  places  cornified  masses  or  whorls  were  apparent.  At 
this  time  we  did  not  consider  blastomycetic  dermatitis,  and  did 
not  look  for  the  organisms  found  in  this  disorder,  though  it  is 
strange  they  should  have  escaped  observation,  as  they  are  numerous 
in  most  of  the  sections  that  have  been  preserved.  The  organisms 
in  their  morphological  features  correspond  closely  with  those 
described  by  Gilchrist  and  others.  The  organisms  are  found 
usually  in  pairs,  both  in  giant  cells  in  the  intra-epitheHal  abscesses 
and  in  the  corium,  but  not  within  other  cells.  Although  these 
bodies  are  present  in  large  numbers,  budding  forms  are  difficult 
to  demonstrate,  partially  because  the  sections  have  faded  some- 
what.    Distinct  budding  forms  are  seen,  however. 

Tissue  was  removed  from  a  very  recent  lesion  on  the  face,  soon 
after  it  had  assumed  the  verrucous  character,  and  used  for  culture 
and  inoculation  experiments  with  negative  results.  A  piece  of 
this  tissue  was  also  hardened  in  alcohol.  Something  over  two 
hundred  sections  were  stained  and  examined  for  bacilli.  In  three 
of  these  sections  (supposed')  tubercle  bacilH  were  found.  In  one 
section  there  was  but  one,  in  another  there  were  two,  while  in  a 
third  there  were  three  fairly  distinct  baciUi.  These  were  all  found, 
however,  in  a  small  abscess  communicating  with  the  surface  of  the 
skin. 

Case  VI. — Clinical  history. — Mrs.  ]\I.  applied  to  Dr.  Hyde  for 
treatment  on  February  12,  1900. 

Patient  is  a  German,  fifty-one  years  old.     Previous  history  good. 
On  the  dorsal  skin  of  the  first  metacarpal  are  two  crusted 

(  ('  Note.  H.  T.  R.)  Hence  if  there  were  tuberck  bacilli,  it  seems  probable  that  their  presence  was 
accidental.     Also  it  is  possible  that  they  may  have  been  saprophytic  acid-proof  bacilli. 

Dr.  Walker,  who  performed  the  autopsy,  and  made  the  diagnosis  of  pulmonary  miliary  tuberculosis, 
recently  re-examined  sections  of  the  lung  tissue,  and  found  the  nodules  to  be  composed  almost  entirely 
of  "blastomycetes,"  and  unassociated  w^ith  tubercle  bacilli.  He  reported  his  findings  at  the  recent  meet- 
ing of  the  American  Dermatological  Society  in  Chicago  (igoi).  The  importance  of  this  finding  will  be 
referred  to  later.] 


Oidiomycosis  of  the  Skin  and  Its  Fungi  97 

ulcers,  one  near  the  proximal,  the  other  over  the  distal  extremity 
of  this  bone.  A  third  lesion  lies  near  the  distal  end  of  the  fifth 
metacarpal. 

The  three  lesions  now  present  are  each  roughly  circular  and 
covered  with  a  thick,  dense,  dirty-yellow,  closely  adherent  crust, 
situated  on  a  prominently  elevated  base.  On  removing  the  crusts, 
bleeding  occurs  and  small  portions  of  tissue  are  torn  away.  The 
exposed  surface  is  verrucous  or  papilHform.  Pus  lies  in  the  clefts 
separating  the  papillae. 

Cultures. — Pus  from  the  base  of  the  ulcer  and  from  the  minute 
abscesses  of  the  areolar  skin  were  studied  in  a  potassium  hydrate 
mount.  Verrucous  tissue  was  teased  and  mounted  in  a  similar 
solution.  A  study  of  the  crusts  in  such  mounts  proved  unsatis- 
factory. 

In  the  pus  and  teased  tissue  were  found  resistant  spherical 
cells,  which  possessed  a  thick  capsule  and  a  central  protoplasm. 
A  few  vacuolated  and  numerous  budding  forms  were  found.  The 
protoplasm  was  finely  granular,  and  usually  there  were  several 
larger,  highly  refractive  bodies.  Several  cells  measured  had 
diameters  of  from  7.5  to  9.4  microns.     A  few  were  oblong. 

February  15,  1900,  ordinary  media  were  inoculated  with  crusts, 
with  pus  from  small  abscesses,  and  with  a  piece  of  diseased  tissue, 
which  had  been  teased  in  sterile  bouillon.  The  crusts  yielded  a 
prolific  growth  of  ordinary  fungi,  forming  greenish  and  brownish 
pigment  and  having  spore-bearing  aerial  hyphae.  Staphylococcus 
aureus  and  albus  occurred  Hberally. 

The  pus  yielded  almost  a  pure  growth  of  a  fungus  in  isolated 
colonies  and  on  all  the  tubes  inoculated.  The  cultures  from  the 
tissue  showed  this  fungus  in  only  one  tube.  A  small  number  of 
colonies  of  staphylococci  developed.  The  staphylococci  were  not 
followed  out  pathogenetically. 

It  required  ten  days  for  the  fungus  to  develop  macroscopically. 
As  the  appearance  of  the  cultures  was  an  unexpected  one,  it  is 
possible  they  may  have  been  discoverable  earlier. 

The  colonies  were  intimately  incorporated  with  the  media 
(agars  and  blood-serum).  The  central  point  was  sKghtly  elevated, 
opaque,  and  grayish  in  color,  and  myceHal  rays  extended  peripher- 


gS  Contributions  to  Medical  Science 

ally,  growing  less  numerous  as  the  border  was  approached.  There 
was  no  growth  above  the  surface.  By  using  a  low  magnification, 
individual  threads  were  visible.  They  shot  out  straight  and  gave 
off  branches  at  an  acute  or  right  angle.  Segmentation  of  the 
mycehum  could  not  be  determined. 

Attempts  to  grow  succeeding  generations  on  all  available 
soUd  and  liquid  media  failed,  both  at  brood-oven  and  room  tem- 
peratures. 

A  successful  hanging-drop  culture  was  obtained  finally  by 
planting  a  portion  of  glucose  agar  culture  (consisting  of  the  soHd 
medium  impregnated  with  the  growth)  in  a  drop  of  glucose  bouillon. 
Growth  proceeded  slowly.  Straight,  translucent,  capsulated 
threads  extended  from  the  agar  fragment  into  the  bouillon,  reached 
the  border,  and,  growing  further,  carried  with  them  sheaths  of 
the  medium.  Branching  at  an  acute  angle  occurred.  Both  the 
main  threads  and  the  branches  divided  into  segments,  which 
showed  little  tendency  to  separate.  There  formed  both  on  the 
threads  and  their  branches  spherical  or  oblong  ascus-like  cells. 
These  commonly  contained  about  eight  highly  refractive  spherules, 
which  were  structureless.  The  spherical  cells  eventually  separated, 
sometimes  before  the  refractive  spherules  were  conspicuous.  Such 
cells  were  seen  to  bud  like  blastomycetes.  Inoculations  from  this 
colony  on  other  media  gave  no  cultures. 

A  smear  preparation  was  made  from  an  agar  culture,  broken 
up  by  teasing,  and  stained  with  Loefiier's  methylene  blue.  It 
showed  a  close  mixture  of  mycehal  threads,  with  oval,  spherical, 
cyKndrical,  and  budding  cells,  the  two  varieties  forming  about 
equal  parts  of  the  mass.  The  threads  varied  from  two  to  four 
microns  in  thickness,  presented  deUcate  capsules,  which  stained 
faintly,  and  a  central  hyalin-Hke  substance,  which  contained 
granules,  spherules,  and  irregular  masses,  staining  deeply  with 
methylene  blue.  They  branched  freely,  and  possessed  numerous 
lateral  conidia  of  various  sizes.  Often  a  thread  terminated  as  a 
large  spherical  or  ovoid  segment.  Frequently  sessile  buds  had 
sprouted  a  mycelial  thread,  which  might  be  delicate  or  coarse. 

The  threads  segmented  freely,  and  in  this  preparation  the 
segments  were  less  coherent  than  in  the  hanging-drop  cultures. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  99 

Portions  of  threads  and  a  few  spherical  cells  were  completely  filled 
with  a  mass  of  deeply  staining  granules  and  spherules. 

The  spherical  and  oval  forms  were  doubly  contoured  and 
possessed  a  central  structure  similar  to  the  mycelium.  Many 
appeared  to  be  degenerating,  only  the  capsules  staining.  They 
were  from  six  to  ten  microns  in  diameter.  A  few  were  seen  to  bud 
(April  22,  1 901).  After  some  months  it  was  possible  again  to 
inoculate  culture  media  with  pus  from  the  miliary  abscesses.  I 
was  successful  this  time,  by  using  a  maltose  agar,  in  cultivating  a 
mould-fungus  which  morphologically  is  identical  with  the  fungus 
obtained  from  Case  VI.  Animal  inoculations  will  be  carried 
out  and  reported  in  the  future. 

HiSTOPATHOLOGY. — A  small  amount  of  blood  lies  on  the  horny 
layer.  The  contour  of  the  surface  is  most  grotesque,  forming 
many  depressions  and  elevations  of  various  sizes  and  shapes.  A 
few  of  these  depressions  lead  to  ruptured  abscesses.  Others  are 
small  and  cup-shaped  and  filled  with  distorted  masses  of  horny 
material,  retaining  a  rough  lamellar  structure  and  showing  numer- 
ous nuclei.  The  horny  layer  varies  in  thickness  and  structure. 
Everywhere  there  is  a  degree  of  loosening  and  desquamation  into 
lamellae  ol  various  thickness.  Nucleation  of  the  deeper  layers  is 
present  irregularly.  In  places  the  horny  layer  is  absent,  the  rete 
being  exposed.  No  stratum  lucidum  is  evident  in  the  diseased 
area. 

The  granular  layer  shows  great  variability  in  occurrence  and 
depth.  It  may  extend  to  a  depth  of  a  dozen  or  fifteen  cells  where 
there  is  epithehal  prohferation,  forming  a  cone-shaped,  granular 
appearance. 

The  rete  presents  the  carcinomatoid  proliferation  which  is 
a  classical  feature  of  the  infection.  There  are  large  blocks  of 
epithelial  cells  connected  with  the  surface  rete,  and  also  columns 
of  varying  thickness  extending  toward  the  subcutaneous  tissue 
from  these  masses.  There  is  an  intercellular  infiltration  of  wander- 
ing cells,  intensified  in  places  to  small  abscess  formation.  Among 
these  cells  the  polymorphonuclear  leukocytes  predominate.  No 
plasma  cells,  eosinophiles,  or  mast-cells  are  seen  in  the  rete. 

The  rete  cells  are  swollen,  granular,  and  the  prickles  are  con- 


loo  Contributions  to  Medical  Science 

spicuous  except  in  areas  of  partial  cornification.  Abnormal 
comification  occurs  in  the  deeper  part  of  the  rete  and  in  the  down- 
growths,  in  isolated  cells,  and  in  groups  of  cells.  There  are  many- 
incipient  whorls,  but  more  are  found  isolated  in  the  cutis.  Isolated 
examples  of  epitheUal  giant  cell  formation  are  found.  Epithehal 
cells,  which  are  otherwise  apparently  normal,  are  found  including 
other  epitheUal  cells  and  polymorphonuclear  leukocytes.  The 
basal  columnar  layer  is  well  formed. 

The  intra-epitheHal  abscesses  are  characteristic.  They  consist 
largely  of  polymorphonuclear  leukocytes,  granular  material, 
and  nuclear  detritus.  In  addition,  many  loosened  epitheUal  ceUs, 
giant  cells,  and  fungus  cells  are  found.  The  epitheUal  ceUs  are 
spherical,  irregular,  or  filamentous.  The  first  type  has  usuaUy 
a  dense,  highly  acidophiUc  protoplasm  and  a  nucleus  which  may 
stain  deeply  or  faintly.  They  sometimes  include  other  epitheUal 
ceUs  or  leukocytes.  The  filamentous  epitheUal  cells  often  resemble 
fungus  threads,  but  in  clearly  stained  specimens  a  flattened  nucleus 
is  usuaUy  demonstrated.  The  giant  cells  within  abscesses  are 
small  and  not  numerous.  The  abscess  wall  is  composed  of  flattened 
epitheUal  ceUs,  which  are  being  loosened  continuaUy  by  the  ceUular 
infiltrate.  OccasionaUy  larger  abscesses  communicate  with  the 
corium. 

The  cutis  is  occupied  by  well-vascularized  granulation  tissue, 
ceUular  infiltrate,  and  small  abscesses.  Many  vessels  have  rather 
thick  walls  and  a  proUferating  endotheUum.  A  few  scattered, 
altered  elastic  fibers  are  found  in  the  inflammatory  area.  Occa- 
sional bundles  of  white  fibrous  tissue  are  found.  With  a  poly- 
chrome methylene  blue  and  eosin  stain,  this  tissue  stains  a  Ught 
blue-green,  while  the  deeper,  healthy  fibers  stain  with  eosin.  Coil 
glands  found  in  the  diseased  area  show  epitheUal  proUferation. 

The  abscesses  are  not  different  from  those  of  other  cases.  They 
contain  occasional  giant  cells  and  the  organism. 

The  infiltrating  cells  are  polymorphonuclear  and  mononuclear 
leukocytes  and  plasma  cells.  The  last-named  have  the  customary 
distribution,  and  a  few  contain  hyalin  spherules.  Giant  cells  are 
smaU.     A  few  are  found  in  the  granulation  tissue. 

The  organisms  are  found  in  the  intra-epitheUal  and  subcu- 


Oidiomycosis  of  the  Skin  and  Its  Fungi  ioi 

taneous  abscesses  and  in  the  surrounding  granulation  tissue.  They 
are  spherical,  sometimes  in  pairs,  or  presenting  an  oblong  cylindrical 
form,  and  are  frequently  seen  in  process  of  budding.  They  have 
not  been  found  in  giant  cells.  In  the  granulation  tissue  the  number 
of  surrounding  leukocytes  is  sometimes  small.  Structurally  they 
possess  a  thick,  hyalin-like  capsule  and  a  central  protoplasm, 
which  is  granular.  Certain  large  granules  stain  well  with  the 
polychrome  methylene  blue.  Vacuoles,  which  may  be  central  or 
eccentric,  single  or  multiple,  are  found  in  occasional  cells.  A 
"clear  space"  is  seen  inconstantly.  Several  degeneration  forms 
have  been  encountered  (containing  no  central  protoplasm). 

The  capsules  are  frequently  fissured.  Careful  searching  of 
appropriately  stained  sections  failed  to  reveal  tubercle  bacilH. 

No  animal  experiments  were  made. 

Case  VII} — CHnical  history. — Patient  is  seventy-three  years 
old;  bom  in  West  Prussia,  where  he  was  a  farmer;  general  history 
fair. 

In  February,  1898,  while  engaged  in  general  labor,  a  pimple 
appeared  on  the  scalp,  about  one  and  one-half  inches  above  the 
left  ear.  Pus  formed  and  discharged.  The  lesion  crusted  and 
extended  gradually,  causing  no  pain,  but  was  tender  on  pressure. 
Slowly  the  disease  reached  its  present  proportions,  tending  to  heal 
in  the  center.  Six  months  ago  a  pimple  appeared  on  the  left 
malar  skin.  This  suppurated,  extended  rapidly,  and  in  two  months 
was  an  inch  in  diameter.  Since  then  it  has  progressed  continu- 
ously. Both  foci  have  discharged  freely.  The  patient's  general 
health  has  been  unaffected. 

Cultures. — Teased  tissue  and  pus  from  the  small  abscesses 
have  been  examined  repeatedly  with  uniform  findings.  A  capsu- 
lated  budding  organism  is  always  present.  It  is  from  eight  to 
sixteen  microns  in  diameter;  has  no  demonstrable  "clear  space"; 
the  protoplasm  is  finely  granular,  and  there  are  in  addition  larger 
granules  of  nearly  uniform  size,  which  move  rapidly  in  the  pro- 
toplasm. Vacuoles  exist  in  many  cells.  After  standing  for  two 
days  in  a  strong  potassium   hydrate   solution,   the  only  change 

•  Cases  VII,  VUI,  and  IX  are  reported  in  Jour.  Boston  Soc.  Med.  Sciences,  igoi,  s.  P-  4S3- 


I02  Contributions  to  Medical  Science 

noted  was  indentation  of  the  capsule  in  some  of  the  cells  (Fig.  12, 
Plate  7). 

In  the  pus  it  is  common  to  find  the  organism  in  groups  of  four 
to  eight.  Many  single  and  paired  forms  have  been  found  filled 
to  bursting  with  refractive  spherical  bodies,  less  than  one-half 
micron  in  diameter.  Some  of  these  cells  were  budding.  Exactly 
similar  bodies  occurred  free  in  groups  of  twenty  to  fifty.  They 
were  not  seen  in  actual  egression  from  the  parasite.  In  mor- 
phology it  would  be  impossible  to  distinguish  them  from  degenera- 
tion products  of  tissue  cells.  A  platinum  loop  of  the  pus  was 
mixed  with  three  or  four  times  its  volume  of  nutrient  bouillon. 
Many  of  these  small  bodies  were  found,  but  during  a  week's  obser- 
vation none  were  found  to  develop  into  the  fungus  cell. 

On  October  16,  1900,  tubes  of  ordinary  media  were  inoculated; 
some  with  pus  from  abscesses  and  others  with  teased  tissue.  Some 
of  the  tubes  inoculated  with  pus  remained  sterile ;  others  had  a  few 
colonies  of  cocci.  The  tubes  inoculated  with  teased  verrucous 
tissue  grew  numerous  colonies  of  cocci  and  bacilli.  No  fungus 
grew.  This  was  repeated  twice  with  similar  results.  Six  plate 
cultures  of  the  pus  were  made  also,  but  the  expected  fungus  did 
not  grow. 

December  i,  1900,  tubes  of  maltose  agar,  prepared  according 
to  Sabouraud's  method  for  the  ring-worm  fungi,  were  inoculated 
with  pus  from  the  abscesses  visible  in  the  reddened  areola.  The 
skin  was  first  cleansed  with  alcohol.  On  December  7  small  colonies 
were  noted  for  the  first  time,  i.e.,  six  days  after  inoculation.  With 
low  magnification  they  were  seen  to  consist  of  radiating  hyphae. 
Examination  of  a  colony  in  water  showed  hyphae  branching  at  an 
acute  angle,  and  possessing  lateral  pedunculated  conidia — the 
"Aaron's  rod"  appearance.  There  were  no  isolated  spherical 
cells.  In  four  days  more  the  colonies  had  grown  rapidly,  rising 
above  the  surface  and  forming  an  irregular,  grayish  mass,  which 
was  closely  adherent  to  the  medium  and  could  be  broken  up  only 
with  considerable  force.  They  rapidly  became  white  and  powdery 
from  the  formation  of  an  aerial  growth.  Within  three  weeks 
hyphae  reached  the  tube  and  grew  up  along  the  glass  surface.  In 
two  months  nearly  the  whole  inner  surface  of  the  tube  was  covered 


Oidiomycosis  of  the  Skin  and  Its  Fungi  103 

with  a  downy  hyphal  growth,  which  could  be  removed  in  soft 
coherent  flakes  with  the  needle. 

Under  the  microscope  this  structure  is  seen  to  consist  of  fine 
branching  mycelial  threads,  which  have  lateral  conidia  and  asco- 
spores.  The  branching  occurs  at,  or  nearly  at,  right  angles  and  is 
monopodial.  Near  the  ends  of  threads,  branching  is  often  at  acute 
angles.  The  main  hyphae  can  be  followed  to  extreme  lengths. 
Neighboring  hyphae  and  their  branches  unite,  and  the  older  portions 
of  threads  are  divided  into  segments,  which  show  no  tendency  to 
separate.  Conidia  develop  principally  on  the  older  portions  of 
threads  and  on  the  lower  branches,  the  growing  ends  being  almost 
devoid  of  them.  They  may  be  sessile  or  pedunculated,  usually 
having  a  delicate  pedicle,  the  protoplasm  of  which  at  first  com- 
municates with  both  that  of  the  conidium  and  the  hypha.  Later 
a  capsular  partition  separates  each  from  the  other.  The  conidia 
multiply  in  situ  by  budding,  so  that  it  is  common  to  find  a  group 
of  four  to  six  at  the  end  of  a  pedicle.  The  commonest  picture, 
however,  is  that  of  a  thread  giving  off  single  pedunculated  conidia, 
arranged  on  alternating  sides  along  the  course  of  the  hypha.  No 
structure  can  be  seen  in  the  conidial  forms.  Ascospores  ( ?)  form 
at  the  ends  of  hyphae  and  the  higher  branches.  Occasionally  the 
end  of  a  thread  is  dilated  into  a  spherical  form,  with  no  visible 
structure.  The  largest  are  twelve  to  fourteen  microns  in  diameter, 
and  the  protoplasm  is  divided  in  a  radiating  manner,  forming 
regular  cone-shaped  segments.  The  number  could  not  be  exactly 
ascertained.  Young  ascal  cells  are  seen  free  and  attached,  con- 
taining from  one  to  several  spores  ( ?) . 

Exactly  similar  growths  occur  when  the  organism  is  transferred 
to  glycerin,  glucose,  plain  agar,  and  blood-serum,  the  first  visible 
colonies  appearing  in  four  to  six  days.  On  none  of  these  media 
is  the  growth  so  pronounced  and  rapid  as  on  maltose  agar.  On 
plain  agar  it  is  rather  meager,  but  there  are  always  aerial  hyphae, 
which  have  the  structure  described  above.  Viewing  the  reversed 
surface,  the  agars  exhibit  a  rich,  even,  golden-brown  color,  like  that 
of  a  well-colored  meerschaum,  becoming  rather  yellowish  near  the 
margins.  The  aerial  growth  on  ox-blood  serum  is  sometimes  very 
marked. 


I04  Contributions  to  Medical  Science 

No  growth  formed  on  highly  acid  media,  as  +io  oxalic 
acid  agar.  Likewise,  prune-decoction  gelatin  constitutes  a  poor 
mediiun. 

Glycerin  agar  plate  cultures. — Some  difficulty  was  experienced 
in  getting  disseminated  colonies,  owing  to  the  nature  of  the  growth. 
A  portion  of  a  bouillon  culture,  however,  was  broken  up  in  a  sterile 
mortar  and  utilized  for  plate  cultures.  After  three  days  colonies 
are  barely  visible,  and  are  not  conspicuous  until  five  days.  At 
the  end  of  a  week  they  are  one  or  two  millimeters  in  diameter, 
roughly  circular,  star-shaped,  feathery,  sending  out  rays  of  varying 
lengths.  The  borders  are  frayed  and  uneven  to  the  unaided  eye. 
With  low  magnification  the  colonies  appear  entirely  hyphal, 
threads  radiating  from  a  dense  central  network,  and  becoming 
sparse  as  the  periphery  is  approached.  They  branch  at  an  acute 
angle.  No  spherical  conidia  are  seen,  but  there  are  many  lateral, 
elongated,  unicellular  shoots.  Segmentation  of  threads  is  indis- 
tinct (Figs.  7,  8).  The  deep  colonies  grow  as  rapidly  as  the  super- 
ficial.    Eventually  aerial  hyphae  develop. 

Potato. — Small  portions  of  infiltrated  agar  cultures  were  smeared 
over  the  surface  of  sterilized  potato.  A  beginning  growth  is  noted 
in  three  days,  and  in  two  days  more  a  white,  downy  surface  has 
formed.  Under  the  microscope  the  aerial  growth  shows  branching 
hyphae,  with  lateral  sessile  and  pedunculated  conidia  and  elongated 
unicellular  offshoots.  New  colonies  very  early  produce  an  aerial 
growth.  After  the  medium  has  dried,  the  surface  of  the  growth  is 
white  and  powdery  and  rubs  off  easily;  but  beneath  this  it  is  hard 
and  some  force  is  required  to  break  it  up.  The  hard  portion  under 
the  microscope  shows  faintly  segmented  threads,  with  occasional 
pedunculated  conidia,  mingled  with  large  numbers  of  spherical 
cells  of  various  sizes.  Many  of  the  latter  are  small  enough  to  be 
considered  detached  conidia,  while  there  are  other  larger  spherical 
cells  (10-12  microns),  many  of  which  are  budding  typically.  The 
ends  of  some  threads  consist  of  a  spherical  segment.  The  spherical 
cells  are  capsulated  and  granular,  but  none  are  seen  forming  the 
characteristically  shaped  spores  observed  on  the  aerial  h^-phae  of 
maltose  agar  cultures. 

Bouillon. — The  growth  in  plain  bouillon  made  with  meat  extract 


Oidiomycosis  of  the  Skin  and  Its  Fungi  105 

is  more  pronounced  than  in  glycerin  bouillon  with  meat  extract. 
In  sugar-free  bouillon  prepared  by  the  Theobald  Smith  method  it 
is  still  more  marked.  Tubes  were  inoculated  either  with  a  mass 
of  the  aerial  growth  or  with  fragments  of  infiltrated  solid  medium. 
The  first  appearance  of  growth  is  a  hazy,  coherent  zone  surrounding 
the  transposed  fragment.  This  increases  in  size,  and  in  about 
ten  days  other  hazy  flakes  appear,  which  have,  no  doubt,  arisen 
from  cells  loosened  from  the  primary  mass.  Eventually  the 
medium  is  largely  occupied  by  such  material,  the  overlying  fluid, 
however,  being  perfectly  clear.  When  the  fluid  is  completely 
filled  or  nearly  evaporated,  aerial  hyphae  shoot  up  from  the  growth 
as  it  lies  adherent  to  the  tube. 

Microscopically  one  sees  a  tangled  mass  of  long,  straight, 
or  slightly  curved  mycelial  threads,  which  interbranch,  and  a 
small  number  of  spherical  cells,  which  multiply  by  budding.  The 
hyphae  vary  in  structure  and  size.  Some  are  only  one  to  two 
microns  thick,  shghtly  tortuous,  branch  freely,  segment  very 
indistinctly  or  not  at  all,  and  possess  a  granular  protoplasm, 
which  in  addition  is  differentiated  into  oval  or  spherical,  slightly 
tinted  bodies,  nearly  the  width  of  the  thread.  Other  hyphae  are 
six  to  eight  microns  in  thickness,  clearly  segmented,  give  off  short 
branches  and  conidia,  and  possess  a  protoplasm  Hke  that  of  the 
finer  threads.  There  are  threads  of  intermediate  sizes.  At  the 
end  of  a  larger  segmented  hypha  a  cluster  of  spherical  cells  may 
form.  The  terminal  segment  of  a  thread  becomes  spherical,  and 
multiplies  by  budding,  the  process  continuing  until  from  ten  to 
thirty  or  forty  cells  accumulate.  The  cluster  eventually  breaks 
up.  These  spherical  cells  in  small  part  again  produce  buds,  but 
largely  develop  into  new  threads.  Each  contains  from  two  to 
four  or  five  spherical,  highly  refractive  granules.  All  these  forms 
possess  the  customary  capsule.  There  are  a  few  short  chains  of 
spherical  cells  (Figs.  7,  Plate  4;  8,  Plate  5;  11,  12,  Plate  6; 
17,  1 8a,  Plate  9). 

On  litmus  milk  growth  is  very  slow;  the  medium  becomes 
slightly  alkaline  without  being  coagulated.  The  growth  hes  on 
the  surface,  where  it  has  an  oily  appearance. 

Owing  to  the  agglutination  of  the  growth  in  all  media,  successful 


io6  Contributions  to  Medical  Science 

stab-cultures  in  agar  and  gelatin  have  not  been  made.  A  hyphal 
growth  occurs  in  gelatin,  however,  without  liquefaction. 

In  hanging  drops  growth  was  very  slow.  Proliferation  took 
place  by  loosely  segmented  hyphae  and  by  budding.  Fine  threads 
did  not  form. 

Saccharine  solutions  were  not  fermented. 

Using  several  maltose  agar  tubes  at  each  inoculation,  the  same 
organism  has  been  cultivated  four  times  in  a  pure  state  from  the 
mihary  abscesses. 

HiSTOPATHOLOGY. — Vcrrucous  tissue  from  the  border  was 
removed,  the  section  including  adjacent  skin.  One  portion  was 
fixed  in  ten  per  cent  formalin,  and  a  second  in  a  mixture  of  Muel- 
ler's fluid  and  formalin.     ParafiBm  imbedding. 

Over  the  surface  hes  a  large  amount  of  homy  debris,  bacteria 
(largely  cocci),  many  red  blood  cells,  and  multitudes  of  leukocytes. 
The  homy  material  often  has  a  lamellar  arrangement,  the  plates 
being  separated  by  leukocytes,  red  blood  cells,  and  bacteria.  A 
large  proportion  of  the  horny  cells  are  nucleated,  although  horny 
masses  are  found  which  have  undergone  almost  complete  granular 
degeneration.  Rarely  a  "  blastomyces  "  is  found  in  the  horny  debris. 
That  part  of  the  homy  layer  which  is  still  attached  is  relatively 
thin.  Masses  of  concentrically  arranged  cells  frequently  lie  in 
depressions  of  the  rete.  Cornification  is  incomplete,  as  shown 
by  the  presence  of  nuclei,  a  granular  eosinophiUc  protoplasm,  and 
the  breadth  of  the  cells.  Flattened  polymorphonuclear  (a  few 
mononuclear)  leukocytes  lie  in  numbers  between  the  lamellae. 
In  one  place  a  mihary  abscess,  containing  a  pair  of  organisms,  is 
found  within  the  horny  layer.  Stratum  lucidum:  Absent.  Stratum 
granulosum:  Present  in  some  places,  absent  in  others.  When 
present  to  a  prominent  or  normal  degree  the  overlying  horny  cells 
approach  the  normal,  being  usually  non-nucleated  and  having  a 
lamellar  formation.  Where  the  granular  layer  is  entirely  absent 
there  is  the  imperfect  cornification  mentioned — nucleated,  granular, 
partially  flattened  cells,  with  thick  walls.  In  the  depths  of  the 
rete,  keratohyalin  granules  are  found  in  the  outer  cells  of  epitheUal 
pearls;  and  occasionally  an  isolated  cell,  or  a  group  of  three  or 
four,  contains  such  granules.    Stratum  mucosum:    The  enormous 


Oidiomycosis  of  the  Skin  and  Its  Fungi  107 

hyperplasia  characteristic  of  the  affection  is  accentuated.  This 
would  be  inferred  from  the  usual  size  of  the  papillae  as  seen  grossly. 
In  the  amount  of  hyperplasia  of  the  rete  this  case  dupHcates  Case 
IV.  Cross-sections  of  deformed  and  enlarged  papillae  and  of 
intra-epithelial  abscesses  are  numerous.  The  cells  adjacent  to 
the  cutis  have  a  protoplasm  which  is  more  granular,  more  chromo- 
philic,  and  of  smaller  dimensions  than  superficial  cells;  the  nuclei 
stain  more  densely  and  exhibit  mitoses.  The  rete  cells  are  imi- 
formly  large,  granular,  finely  vesiculated,  and  have  distinct  prickles. 
The  interepithelial  spaces  are  often  large.  Except  where  there  are 
small  foci  of  necrosis  the  nuclei  stain  well.  There  are  various  sized 
epithelial  whorls  and  examples  of  premature  and  partial  comifica- 
tion.  In  the  interepithehal  spaces  are  polymorphonuclear  leu- 
kocytes, a  few  eosinophiles  and  mast-cells,  and  red  blood  cells. 
Rarely  a  parasite  is  foimd  in  this  position  (Fig.  3,  Plate  3).  A 
few  large  hemorrhages  are  seen  in  the  rete,  the  surrounding  cells 
being  flattened. 

Intra-epithelial  abscesses  are  numerous  and  variable  in  size. 
They  have  the  usual  contents,  including  giant  cells  and  the  organ- 
isms. 

There  are  many  examples  of  epithelial  giant  cell  formation  in 
the  body  of  the  rete  apart  from  those  in  abscesses.  They  may 
contain  leukocytes  or  other  organisms. 

The  cutis  is  marked  by  greatly  deformed  papillae,  dilated 
blood-  and  lymph- vessels,  and  numerous  small  hemorrhages.  A 
mucoid  appearance  in  the  superficial  cutis  seems  to  have  been 
caused  by  the  marked  edema.  The  fixed  tissue  cells  have  pro- 
liferated excessively.  Plasma  cells  are  less  crowded  than  in  some 
previous  cases.  They  occur  in  groups  and  in  columns,  surrounding 
blood-vessels,  and  occasionally  contain  hyalin  bodies.  Mast-cells 
are  seen  singly  and  in  groups.  Eosinophiles  are  found  in  large 
numbers,  often  in  groups,  constituting  another  point  of  resemblance 
to  Case  IV. 

There  are  tubercles  resembhng  those  of  tuberculosis.  They 
differ  from  the  latter  in  being  well  vascularized.  Giant  cells 
are  exceedingly  numerous,  but  no  tubercle  bacilli  can  be  found. 
The  subepithelial  abscesses  are  like  those  of  other  cases.     Poly- 


io8  Contributions  to  Medical  Science 

morphonuclear  and  mononuclear  leukocytes  infiltrate  the  cutis 
diffusely. 

The  organism  in  tissue. — In  the  cutis  it  is  seen  in  abscesses, 
among  connective  tissue  apart  from  abscesses,  and  in  giant  cells. 
In  the  epithehum  it  occurs  in  the  abscesses  and  between  epithelial 
cells,  often  with  httle  or  no  surrounding  zone  of  leukocytes.  It 
is  found  within  giant  epitheHal  cells,  but  not  within  mononuclear 
cells.  In  all  tissues  it  multiplies  by  the  characteristic  budding 
process.  Groups  of  several  organisms  are  seen  occasionally,  as 
in  Case  IV.  Such  groups  commonly  have  a  small  amount  of  inter- 
cellular cement  substance. 

The  usual  structure  is  found,  a  doubly  contoured  capsule,  a 
clear  zone,  and  a  central  protoplasm.  Polychromatic  methylene 
blue  stains  the  organism  characteristically.  The  capsule  appears 
deep  blue,  the  clear  zone  has  a  reflected  blue  tint,  and  the  central 
protoplasm  stains  reddish-blue.  The  same  results  are  obtained 
with  carbol-toluidin  blue  and  carbol-thionin.  These  stains  make 
the  organism  stand  out  clearly,  while  hematoxylin,  with  eosin 
and  carbol-fuchsin  as  counter-stain,  is  less  satisfactory.  The 
central  protoplasm  is  granular,  sometimes  containing  the  spherical 
granules  of  uniform  size  mentioned  under  preceding  cases.  They 
are  rather  difficult  to  see  in  the  methylene  blue  stains,  as  the  whole 
protoplasm  may  be  deeply  stained.  A  hematoxylin  stain  brings 
them  out  more  clearly.  Vacuoles  are  common.  Degeneration 
forms  are  found  in  numbers,  particularly  within  giant  cells.  Organ- 
isms with  fractured  and  others  with  indented  capsules  are  met 
with  occasionally.     The  average  diameter  is  ten  to  twelve  microns. 

Animal  Experiments. — Guinea-pigs.  i.  Inoculation  of  tissue  beneath  skin 
over  the  right  shoulder.  The  wound  became  swollen,  and  on  the  third  day  death 
resulted.     Staphylococci  cultivated  from  the  wound  and  the  viscera. 

2.  The  coherent  fungus  mass  from  a  bouillon  tube  was  placed  subcutaneously. 
After  three  days  a  nodule  formed,  which  discharged  in  a  week.  In  the  pus  short 
mycelial  forms  were  found.  The  neighboring  lymphatics  continue  enlarged,  but 
the  wound  healed  completely. 

White  Rats.  i.  A  block  of  infiltrated  agar  was  inserted  subcutaneously  with  no 
result. 

Dogs.  I.  Blocks  of  infiltrated  solid  media  were  placed  within  the  abdomen. 
No  observable  effect  after  two  months. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  109 

2.  Intravenous  inoculation  of  a  bouillon  culture  broken  up  in  a  mortar.  After 
ten  days  became  somewhat  emaciated,  and  had  a  purulent  conjunctivitis,  con- 
taining only  cocci. 

One  month  later  the  right  cornea  was  involved  in  a  scar  and  slightly  deformed. 
As  emaciation  and  weakness  were  extreme,  and  the  animal  was  dyspneic,  chloroform 
was  administered.  At  the  autopsy,  all  organs  are  found  to  be  pale  and  otherwise 
unchanged,  except  the  lungs.  Both  parietal  and  visceral  pleurae  are  smooth,  but  the 
latter  is  mottled  gray  and  red,  and  presents  rather  large  grayish  elevations.  Only 
portions  of  the  upper  lobes  crepitate,  the  remaining  lung  tissue  being  involved  in  a 
rather  soft  nodular  consolidation.  Section  shows  numerous  grayish,  confluent, 
and  ill-defined  nodules,  from  which  fluid  containing  minute  granules  can  be  squeezed. 
A  similar  fluid  escapes  from  the  bronchi.  No  scars  or  well-marked  abscesses  are  noted. 
A  portion  of  a  nodule  teased  and  examined  in  water  shows  almost  a  solid  mass  of 
spherical,  capsulated,  and  budding  organisms,  which  are  often  filled  with  refractive, 
spore-like  bodies;  similar  bodies  are  seen  singly  and  in  groups;  extracellular  organ- 
isms may  have  a  marked  adventitious  capsule,  are  often  agglutinated,  and  one  or 
more  may  exist  in  a  mono-  or  multi-nuclear  tissue  cell. 

Apparently  gradation  forms  exist  between  the  extracellular  spore-like  bodies 
and  distinctly  capsulated  forms  of  the  organism,  but  as  these  bodies  mounted  in  a 
hanging  drop  do  not  develop  further,  their  position  as  spores  remains  uncertain. 
The  only  clear  method  of  proliferation  in  the  lung  tissue  is  by  budding.  Vibrating 
granules  are  often  found  in  the  fungus  cells.  The  organisms  in  the  bronchial  fluid 
have  a  similar  morphology. 

Some  fluid  from  a  nodule,  mounted  in  a  hanging  drop  of  bouillon,  showed  in  a  few 
hours  the  sprouting  of  hyphae  from  the  spherical  cells,  both  those  containing  spore- 
like bodies  and  those  with  relatively  clear  protoplasm.  Within  twenty-four  hours 
nearly  all  cells  had  started  similar  processes,  which  eventually  reached  great  lengths, 
became  segmented,  and  gave  off  lateral  conidia  and  unicellular  offshoots.  The 
spore-like  bodies  took  no  evident  part  in  this  development,  and  they  disappeared 
largely  after  a  few  days. 

Pure  growths  of  the  fungus  inoculated  were  obtained  from  lung  nodules,  but 
inoculations  from  other  viscera  and  the  blood  remained  sterile. 

A  lung  nodule  sectioned  and  stained  consists  almost  entirely  of  fungous  cells, 
with  a  doubly  contoured  capsule,  clear  space,  and  protoplasm,  which  often  contains 
stained  spore-like  bodies  and  vacuoles.  They  are  often  in  groups,  surrounded  and 
held  together  by  a  granular,  cement  substance.  Mono-  and  multi-nuclear  endothelial 
cells  contain  one  or  more  organisms.  Remnants  of  alveolar  structure  are  seen,  but 
the  cells  are  very  granular.  Vessels  are  moderately  congested;  no  hemorrhages. 
There  are  small  masses  of  fibrin  and  a  few  mast-cells. 

The  reaction  to  Gram's  stain  is  peculiar,  as  only  one  out  of  many  dozen  cells  is 
stained,  and  very  deeply;  the  capsule  is  partly  decolorized. 

Extracellular  spore-like  bodies  stain  with  nuclear  dyes,  but  they  present  no 
differentiated  structure.  Organisms  are  not  found  in  peribronchial  lymph  glands. 
Kidneys:  Diffuse  granular  degeneration  of  the  epithelium;  ,a  few  foci  of  perivascular 
plasma  cell  infiltration  and  endothelial  proliferation;  no  fungous  cells.  Liver:  Fatty 
degeneration.     Brain,  spleen,  heart,  and  suprarenals  normal. 

Rabbit.    Intravenous  inoculations  of  bouillon  culture.     No  effect. 


no  Contributions  to  Medical  Science 

Summary  of  the  Biological  Features  of  the  Organism. — 
In  tissues  it  proliferates  only  by  budding,  so  far  as  can  be  observed, 
although  certain  granules  in  the  cells  and  the  location  of  large  forms 
between  epithelial  cells  suggest  that  spores  may  form  and  be 
transported  by  lymph  currents. 

From  three  to  eight  days  are  required  for  development  on 
artificial  media.  The  medium  becomes  infiltrated  with  h^-phae 
and  an  aerial  growth  occurs,  on  the  threads  of  which  pedunculated 
lateral  conidia  and  terminal  ascus  cells  form,  which  contain  spore- 
like bodies.  Aerial  threads  are  divided  into  closely  cohering 
segments.  Conidia  may  multiply  by  budding.  The  buried 
hyphae  are  more  loosely  segmented  and  have  few  lateral  conidia, 
but  many  unicellular  shoots;  some  budding  cells  are  seen  also. 
In  Hquid  media  the  growth  is  mycelial,  branching,  segmented,  few 
lateral  conidia,  but  terminal  spore  groups,  the  elements  of  which 
multiply  by  budding.  Milk  is  coagulated;  produces  a  slight 
alkahne  reaction.  Gelatin  is  not  liquefied.  On  plates  feathery, 
irregular,  hyphal  colonies  develop,  the  submerged  growing  as  well 
as  the  surface  colonies.  On  all  media  the  protoplasm  is  clear  or 
finely  granular,  capsulated,  and  may  contain  spherical  or  oval, 
sHghtly  tinted  bodies  of  uncertain  function.  Few  vacuolated 
bodies  are  seen. 

ProHferation  then  takes  place  by: 

1.  Lateral  aerial  conidial  formation. 

2.  Terminal  ascospores  (?). 

3.  Separation  of  mycelial  segments. 

4.  Budding  of  terminal  spore  groups. 

Case  VIII .^ — Mrs.  A.  M.,  age  twenty-eight;  Bohemian;  occu- 
pation, housework.  Has  had  the  usual  diseases  of  childhood. 
No  specific  history. 

On  examination  the  disease,  which  first  appeared  about  twenty 
months  ago,  was  seen  to  involve  the  entire  region  surrounding 
the  right  orbit,  extending  well  over  the  cheek  and  up  on  to  the 

'  Occurred  in  the  service  of  Dr.  W.  L.  Noble  at  the  Illinois  Eye  and  Ear  Infirmary;  it  was  referred 
to  Professor  Montgomery  for  diagnosis.  I  am  indebted  to  them  for  the  privilege  of  reporting  the  case. 
Professor  Montgomery  made  a  rejxirt  of  the  case  before  the  American  Dermatological  Association,  May 
30,  1901.    See  also  note  to  Case  \^II. 


Oidiomycosis  of  the  Skin  and  Its  Fungi       •     iii 

forehead.  The  process  had  travelled  over  the  bridge  of  the  nose 
and  had  involved  the  left  upper  eyelid  and  a  small  region  imme- 
diately below  the  eye.  The  clinical  features  were  identical  with 
those  of  several  cases  of  blastomyces  previously  described. 

Cultures. — ^At  first  no  fungus  grew  on  media  inoculated  with 
tissue  and  surface  pus ;  ordinary  bacteria  appeared  freely. 

Later  the  skin  overlying  several  myxomatous  tubercles  was 
steriUzed  with  alcohol,  incised,  and  the  soft  contents  scraped  out 
with  a  sterile  curette.  Examination  of  the  fresh  scrapings  showed 
groups,  pairs,  and  single  forms  of  spherical,  capsulated,  budding 
cells. 

Inoculations  of  this  tissue  were  made  on  ordinary  media  and 
maltose  agar.  A  growth  was  not  noted  for  five  or  six  days.  Then 
it  was  seen  that  hyphae  had  sprouted  from  fragments  of  tissue 
placed  on  maltose  agar,  but  no  growth  occurred  on  other  media. 
In  about  two  weeks  hyphae  rose  above  the  surface,  and  in  a  week 
more  the  mould-fungus  nature  of  the  organism  was  clear. 

In  morphology  and  methods  of  proliferation  this  fungus  appears 
identical  with  those  cultivated  from  Cases  VI,  VII,  IX,  and  X. 

Animal  inoculations  are  in  progress. 

HiSTOPATHOLOGY. — The  tissue  removed  included  a  myxomatous 
nodule  and  a  small  amount  of  the  verrucous  border.  So  much 
healing  had  occurred  under  the  administration  of  the  iodide  of 
potassium  that  the  typical  findings  could  not  be  anticipated. 
In  KOH  mounts  of  fresh  tissue,  groups  of  blastomycetoid  bodies 
were  found.  EpitheHal  hyperplasia  is  marked  only  in  the  verru- 
cous tissue,  the  epidermis  covering  the  nodule  being  only  moder- 
ately thickened,  and  not  sending  prolongations  into  the  underlying 
tissue.  Only  a  few  intra-epithelial  abscesses  are  encountered  in 
the  verrucous  tissue  and  none  in  the  covering  of  the  nodule;  three 
are  found  in  the  corium.  There  is  moderate  diffuse  infiltration 
of  the  rete  in  the  verrucous  tissue,  with  leukocytes,  but  none  in 
that  of  the  nodule. 

The  infiltrating  cells  of  the  corium,  as  in  other  cases,  are  poly- 
morpho-  and  mono-nuclear  leukocytes,  and  plasma  cells;  in  addi- 
tion, there  are  numerous  eosinophiles,  as  in  Cases  VII  and  IX, 
There  are  many  giant  cells  of  the  tuberculous  type,  and  a  number 


112  Contributions  to  Medical  Science 

of  cell  nodules  are  present.  One  of  these  is  very  similar  to  the 
t}T)ical  tubercle;  however,  there  is  no  central  necrosis,  and  lym- 
phocytes infiltrated  the  structure  diffusely. 

There  is  a  large  amount  of  cicatricial  tissue,  corresponding  to 
the  degree  of  healing  noted  cHnically. 

Few  fungous  cells  are  found.  They  lie  in  two  groups,  each 
surrounded  by  leukocytes.  The  individuals  measure  about  ten 
microns  in  diameter,  proHferate  by  budding,  contain  spore-Uke 
bodies,  possess  a  doubly  contoured  and  a  thin  adventitious  capsule. 

Case  /X.'— Clinical  history.— Mrs.  J.  K.,  age  thirty-three  years, 
born  in  Germany.  She  had  poor  health  prior  to  puberty,  and 
passed  through  the  usual  diseases  of  childhood.  Close  questioning 
reveals  no  history  of  syphihs. 

A  Httle  more  than  two  years  ago  three  pustules  appeared  at 
about  the  same  time,  one  in  the  skin  overlying  the  left  lower  jaw, 
another  on  the  dorsum  of  the  left  wrist,  and  the  third  on  the  right 
buttock.  The  one  on  the  face  continued  to  spread  at  all  points, 
became  more  and  more  warty,  and  discharged  pus  freely. 

The  diseased  area  was  excised  by  Dr.  Graham,  a  zone  of  healthy 
skin  one  and  a  half  centimeters  wide,  and  the  underlying  fatty 
tissue,  being  included. 

Gross  Appearances. — The  principal  diameters  of  the  area 
are  5.5  by  4.5  centimeters,  the  form  being  roughly  ovoid,  and  the 
contour  a  little  irregular.  The  lesion  presents  a  central  and  a 
peripheral  portion.  The  latter  is  from  0.5  to  2.0  centimeters  wide, 
elevated  from  0.2  to  i.o  centimeters  above  the  surrounding  skin, 
and  has  a  characteristic  coarse  verrucous  or  papillomatous  appear- 
ance. It  meets  the  surrounding  skin  abruptly,  and  where  it  is 
thickest  and  highest  becomes  rounded  and  overhangs  and  conceals 
the  healthy  border,  cauliflower-hke.  The  papillae  are  of  various 
sizes  and  grotesque  shapes.  Some  are  millet-seed  size  and  thickly 
set,  each  being  separated  from  its  neighbor  by  a  deep,  narrow 
cleft;  this  type  occurs  nearest  the  central  portion.  Others  are 
large  fungous  masses,  from  0.2  to  0.5  centimeters  in  thickness, 
with  a  relatively  slender  base,  and  having  a  surface  set  with  second- 

»  See  note  to  Case  VII. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  113 

ary  millet-seed  size  or  larger  papillae;  these  occur  principally  in 
the  outer  part  of  the  peripheral  zone.  In  general,  the  papillae  are 
shorter  and  simpler  as  the  center  of  the  lesion  is  approached  (Fig. 
lb,  Plate  i).  The  central  portion  is  from  0.5  to  2.0  centimeters 
wide  and  3.0  centimeters  long.  It  is  depressed  about  0.5  centi- 
meter below  the  level  of  the  highest  peripheral  portion,  but  is 
elevated  sUghtly  above  that  of  the  surrounding  skin.  Its  surface 
is  relatively  smooth,  and  at  a  few  points  somewhat  glistening. 
An  outer  zone,  however,  is  coarsely  granular,  this  appearance  being 
caused  by  inconspicuous,  blunt,  short  papillae.  The  papillomatous 
tissue  is  exceedingly  friable  and  was  torn  shghtly  in  manipulation. 
The  central  area  is  a  little  more  dense,  and  suggests  new  cicatricial 
tissue.  The  whole  lesion  is  more  or  less  covered  with  blood, 
secondary  to  the  operation. 

The  tissue  was  bisected  to  expose  the  deeper  structure.  The 
subcutaneous  fat  is  sharply  and  rather  evenly  separated  from  the 
overlying  diseased  tissue,  and  presents  a  few  moderately  enlarged 
vessels.  Between  the  subcutaneous  fat  and  the  papillary  layer 
is  a  homogeneous,  whitish  zone,  from  0.2  to  0.5  cm.  deep,  which 
in  places  exhibits  hemorrhages,  varying  from  pin-point  size  to 
several  milHmeters  in  extent.  Some  have  an  oblong  or  irregular 
shape.  This  zone,  no  doubt,  represents  the  densely  infiltrated 
corium.  The  papillary  layer  consists  of  two  portions:  a  surface 
zone,  represented  by  shaft-Hke  papillae,  separated  from  each  other 
by  clefts;  and  a  buried  zone,  in  which  the  papillae  are  united  side 
by  side.  Those  in  the  latter  zone  are  sharply  contoured  by  white 
lines  of  horny  tissue,  which  caps  the  surface,  sheaths  the  deeper 
portion,  and  cements  each  papilla  to  those  adjacent  for  the  depth 
of  0.2  to  0.4  cm.  The  extension  of  these  papillae  above  the  surface 
of  course  gives  rise  to  the  isolated  surface  papillae.  A  central 
"core"  of  vascular  tissue  occupies  the  center  of  each  papilla. 

It  is  not  possible  in  any  portion  of  the  tissue  to  detect  microscopic 
abscesses ;  this  probably  is  due  to  an  unfortunate  accident  in  tech- 
nique. (However,  after  the  tissue  had  been  passed  through  the 
KaiserHng  solutions  for  preservation  as  museum  specimens,  minute 
abscesses  were  seen  in  cross-sections.) 

HiSTOPATHOLOGY. — Two    or    three   papillae   were   clipped   off 


114  Contributions  to  Medical  Science 

and  examined  in  a  KOH  solution.  Portions  of  elastic  fibers, 
epithelial,  blood,  and  other  tissue  cells  were  recognized  readily. 
No  difficulty  was  encountered  in  finding  numbers  of  a  blastomyces- 
like  organism.  Eight  occurred  in  one  field;  many  other  fields, 
however,  contained  none. 

As  to  form,  they  were,  as  usual,  spherical,  budding,  and  occa- 
sionally elongated;  a  few  cup-shaped  and  sickle-forms  were  seen. 
Several  groups  of  three  to  five  organisms  each  were  found.  The 
capsule  appeared,  as  usual,  with  a  clear  double  contour  and  of 
uniform  thickness.  The  protoplasm  in  certain  cells  was  almost 
clear,  in  others  contained  a  few  granules,  and  in  many  was  com- 
pletely occupied  by  minute  spherical  refractive  bodies,  smaller 
than  those  ordinarily  seen  in  Cases  VI,  VII,  and  X.  Transition 
forms  between  the  minute  bodies  and  the  adult  capsulated  cell  are 
present.  They  are  intermediate  in  size,  and  present  no  differen- 
tiation except  a  very  delicate  capsule.  Two  cells  were  found 
enclosing  pigment  granules.  The  average  diameter  of  twenty 
cells  was  9.5  microns. 

Four  portions  of  tissue  have  been  used  for  microscopic  study: 
(i)  The  surrounding  relatively  healthy  skin;  (2)  the  outer  densely 
verrucous  zone;  (3)  the  central  smooth,  cicatrized  portion;  and 
(4)  a  point  intermediate  between  2  and  3.  All  tissues  had  similar 
treatment,  being  in  Zenker's  fluid  for  a  few  seconds  by  accident, 
washed  in  water  for  two  hours,  fixed  in  formalin  and  water,  dehy- 
drated, cleared  in  cedar  oil,  and  imbedded  in  paraffin. 

The  description  of  the  histological  pathology  of  Case  VII 
applies  in  all  essential  details  to  that  of  this  one.  More  underlying 
tissue  is  included  in  this  case,  which  merely  shows  that  the  leu- 
kocytic cell  infiltrate  extends  superficially  into  the  subcutaneous 
fatty  and  muscular  tissue.  The  number  of  intra-epithelial  and 
subcutaneous  abscesses  is  large.  The  organisms  occur  singly,  in 
pairs,  and  in  groups,  are  occasionally  surrounded  by  an  adventitious 
capsule,  may  contain  staining  spore-like  bodies  and  vacuoles,  and 
possess  a  doubly  contoured  membrana  propria;  they  are  found 
in  all  abscesses,  between  relatively  healthy  epithelial  cells,  and  in 
the  granulation  tissue  of  the  corium.  As  in  Case  VII,  eosinophiles 
are  a  conspicuous  feature  of  the  cell  infiltrate,  extending  markedly 


Oidiomycosis  of  the  Skin  and  Its  Fungi  115 

into  the  rete  and  intra-epithelial  abscesses.  The  rete  shows  the 
types  of  atypical  cornification,  and  other  epithehal  changes  noted 
in  other  cases. 

Plasma  cells  are  numerous,  but  polymorphonuclear  leukocytes 
are  equally  so. 

The  areolar  skin  shows  a  small  amount  of  cell  infiltrate  and  a 
few  minute  abscesses  in  the  corium. 

In  the  central  depressed  portion  of  the  lesion  the  surface  papillae 
have  disappeared  almost  entirely,  but  the  subcutaneous  changes 
are  as  pronounced  as  in  any  portion  of  the  tissue. 

There  are  remnants  of  elastic  tissue  in  the  infiltrated  corium. 
Many  groups  of  hyalin  bodies  are  found  in  plasma  cells.  Giant 
cells  in  the  rete  and  corium  are  numerous  and  of  the  tuberculous 
type.     Many  contain  the  fungus. 

Cultures. — A  portion  of  verrucous  tissue  and  infiltrated  corium, 
with  a  small  amount  of  bouillon,  was  placed  in  a  sterile  mortar 
and  thoroughly  disintegrated.  This  suspension  was  used  in 
inoculating  various  media,  including  maltose  agar;  plate  cultures 
also  were  made  in  plain-,  glycerin-,  and  glucose-agar.  The  tubes 
and  plates  were  examined  daily  under  a  low  objective.  On  the 
fourth  day  hyphae  began  to  grow  out  from  several  tissue  fragments. 
Growth  proceeded  slowly,  the  threads  at  first  growing  on  the 
surface  or  immediately  beneath  it.  In  eight  or  ten  days  an  aerial 
growth  was  perceptible ;  this  increased  and  soon  appeared  identical 
with  the  organism  cultivated  from  Case  VII.  Study  of  its  mor- 
phology and  methods  of  proliferation  also  shows  the  apparent 
identity  of  this  fungus  with  that  from  Case  VII.  That  is,  the 
aerial  hyphae  bear  lateral  pedunculated  conidia,  which  may  multi- 
ply by  budding,  and  terminal  bodies,  which  seem  to  contain  spores; 
there  are  large  cells  filled  with  refractive,  structureless,  spore-like 
bodies;  submerged  spherical  cells  multiply  by  budding,  and  the 
submerged  mycelium  is  segmented,  contains  nucleus-like  bodies, 
and  gives  off  conidia  and  unicellular  shoots.  Both  aerial  and 
submerged  hyphae  present  monopodial  branching. 

Animal  Experiment. —  In  order  to  eliminate  the  possibility  of  tuberculosis, 
a  portion  of  tissue  was  placed  subcutaneously  over  the  inguinal  lymph  glands  of  a 
guinea-pig.  Swelling  was  noted  in  a  few  days,  and  a  hard  nodule  soon  formed,  from 
which  thick,  creamy  pus  could  be  pressed.     The  latter  contained  spherical  forms,  the 


ii6  Contributions  to  Medical  Science 

protoplasm  of  which  was  often  differentiated  into  small,  spherical,  refractive  bodies. 
Inoculations  on  media  resulted  in  growths  of  a  fungus  which  morphologically  is  iden- 
tical with  that  cultivated  from  the  tissue.  In  the  pus  the  organism  prohferated  by 
budding.     The  inoculation  wound  healed  gradually,  no  general  infection  resulting. 

Case  X. — This  case  was  brought  to  my  notice  through  the 
kindness  of  Dr.  W.  E.  Marquardt,  who  has  furnished  the  following 
clinical  history. 

Mr.  R.,  fifty-eight  years  old,  a  carpenter,  was  bom  in  Germany; 
general  history  good. 

His  affection  began  in  December,  1895,  as  a  papule  half  an  inch 
above  the  styloid  process  of  the  left  radius. 

On  examination  it  presents  a  reddish  granular  surface,  excavated 
in  places  and  surrounded  by  an  elevated,  reddish,  puffy  areola. 
Throughout  the  lesion  are  pin-head-sized  abscesses,  which  are 
more  densely  distributed  at  the  margins. 

As  the  patient  would  not  consent  to  the  removal  of  tissue  for 
examination,  a  report  on  the  histopathology  cannot  be  given. 

Cultures. — When  first  seen  with  Dr.  Marquardt,  the  lesion 
had  been  macerated  in  moist  dressings,  and  no  miliary  abscesses 
were  visible.  Papillae  were  cKpped  off,  however,  and  inoculated 
on  ordinary  media  and  maltose  agar.  At  the  same  time  a  few 
papillae  teased  and  mounted  in  a  KOH  solution  showed  capsulated, 
budding  cells,  existing  singly,  in  pairs,  and  in  groups.  Many  were 
filled  with  structureless,  spherical,  refractive  bodies,  Uke  those 
seen  in  some  previous  organisms.  The  growth  of  ordinary  bacteria 
on  all  tubes  was  so  abundant  that  new  inoculations  were  tried. 
The  patient's  hand  had  been  dressed  dry  for  a  number  of  days,  and 
many  miliary  abscesses  had  formed.  Dr.  Marquardt  kindly  inocu- 
lated tubes  of  maltose  agar  with  pus  from  the  abscesses,  after 
cleansing  the  skin  with  alcohol,  and  sent  them  to  the  laboratory. 
A  week  later  the  development  of  a  mould-fungus  had  begun.  It 
is  necessary  to  add  only  that  this  fungus  in  its  morphology  and 
reproduction  appears  identical  with  those  cultivated  from  Cases 
VI,  VII,  VIII,  and  IX.  The  organism  was  obtained  so  recently 
that  it  has  not  been  possible  to  terminate  animal  experiments. 

Case  XI. — This  was  a  bottle  of  celloidin  sections,  with  an 
absolutely    imknown    history,    bearing    the    label    "Tuberculosis 


Oidiomycosis  of  the  Skin  and  Its  Fungi  117 

verrucosa  cutis."  It  was  one  of  a  number  of  cases  of  supposed 
tuberculosis  of  the  skin  kindly  placed  at  my  disposal  by  Dr. 
Le  Count.  The  sections  are  all  quite  thick.  The  method  of  fixation 
is  not  known. 

The  horny  layer  is  thickened,  desquamating,  imperfectly  corni- 
fied,  and  infiltrated  moderately  with  polymorphonuclear  leukocytes. 

In  places  the  granular  layer  is  thickened,  and  it  is  absent  in 
others. 

The  rete  is  exposed  occasionally,  and  a  sinus  is  seen  communicat- 
ing with  an  abscess  in  the  corium. 

There  is  dense  infiltration  of  the  rete  with  polymorphonuclear 
leukocytes.  This  layer  also  presents  the  great  hyperplasia  and 
deformity  characteristic  of  the  disease,  and  contains  many  miliary 
abscesses  of  various  sizes.  The  cells  are  large,  vesicular;  the 
nuclei  usually  stain  well,  and  the  prickles  are  distinct  except 
around  abscesses  and  areas  of  abnormal  cornification.  There  are 
occasional  examples  of  epithelial  giant  cell  formation,  and  of  the 
inclusion  of  leukocytes  by  epithelial  cells  otherwise  apparently 
normal.  The  basal  columnar  formation  is  maintained  with  some 
deformities,  the  protoplasm  staining  deeply.  No  mitoses  are 
found  (perhaps  because  of  the  method  of  fixation).  Large  knob- 
like masses  of  rete  cells  are  quite  sure  to  contain  one  or  more 
minute  abscesses. 

The  cutis  is  largely  a  mass  of  granulation  tissue,  densely  infil- 
trated with  wandering  cells,  of  which  polymorphonuclear  leukocytes 
form  the  preponderating  number.  Lymphocytes  also  occur  in 
great  numbers,  having  a  fairly  uniform  distribution.  Frequently 
the  polymorphonuclears  form  distinct  abscesses.  Plasma  cells 
are  numerous  and  have  the  characteristic  distribution  for  this  cell. 
HyaHn  bodies  have  not  been  found  in  them  nor  in  other  portions 
of  this  tissue.  Mast-cells  cannot  be  demonstrated.  This  again 
may  be  due  to  fixation  methods  or  the  age  or  method  of  preservation 
of  the  tissue.  There  are  only  fragments  of  poorly  staining  elastic 
tissue  in  the  parts  severely  involved.  Hair  foHicles,  sebaceous  and 
sudoriparous  glands  are  included  in  the  sections.  The  hair  and 
sebaceous  glands  are  normal,  but  surrounded  by  an  excess  of 
fibrous  tissue.     The  sweat  glands  also  are  surrounded  by  an  excess 


ii8  Contributions  to  Medical  Science 

of  connective  tissue,  many  of  the  tubules  are  almost  obliterated, 
and  others  are  completely  filled  with  epithelial  cells.  The  number 
of  giant  cells  in  the  corium  is  strikingly  small.  In  the  intra-epithe- 
lial  abscesses  there  is  a  large  number  of  detached  and  alterated 
epithehal  cells.  They  possess  the  characters  described  in  connec- 
tion with  other  cases  of  this  series. 

The  organism  is  found  in  the  abscesses  of  the  epithelium  and 
cutis,  and  in  the  granulation  tissue  of  the  latter.  The  large  number 
in  the  subepithelial  tissue  is  a  distinguishing  feature  of  the  case. 
The  diameter  is  from  ten  to  sixteen  microns.  There  are  many 
budding  and  some  large  cylinder-like  forms.  As  in  other  cases, 
the  organism  possesses  a  doubly  contoured  capsule  and  a  central 
protoplasm,  which  is  both  finely  and  coarsely  granular.  A  number 
of  coarse  spherical  granules  are  sometimes  arranged  peripherally 
in  the  protoplasm. 

Tubercle  bacilli  could  not  be  found. 

Case  XII. — This  patient,  Mr.  R.,  was  admitted  into  the  Presby- 
terian Hospital  in  the  service  of  Professor  Senn.  The  history  is, 
unfortunately,  meager.  He  was  forty  years  old  and  married. 
His  personal  history  was  marked  "good."  He  noticed  a  soreness 
of  the  right  hamstring  muscles  in  January,  1897.  ^^  September 
12,  1899,  a  piece  of  tissue  was  removed  without  anesthesia.  The 
clinical  diagnosis  was  "tuberculosis  of  knee-joint." 

Subsequent  attempts  to  locate  the  patient  were  futile.  The 
tissue  in  the  possession  of  the  laboratory  is  supposedly  that  removed 
at  the  last  operation  recorded.  Professor  Hektoen  discovered  the 
nature  of  the  tissue,  and  kindly  submitted  sections  for  the  purpose 
of  this  work. 

The  characteristic  changes  of  blastomycetic  dermatitis  are 
found  throughout  the  sections  both  in  the  epithelium  and  sub- 
epithelial tissue.  To  enumerate:  There  are  the  great  epithelial 
hyperplasia,  leukocytic  infiltration  of  all  layers  of  the  epithelium, 
intra-epithelial  abscesses,  the  usual  dense  infiltration,  congestion, 
and  edema  of  the  cutis.  Abscesses  are  found  in  the  cutis,  and 
plasma  cells  are  numerous.  The  organisms  are  found  in  the  intra- 
epithelial and  subepithelial  abscesses  and  in  the  granulation  tissue 


Oidiomycosis  of  the  Skin  and  Its  Fungi  119 

of  the  cutis.  They  are  spherical,  and  budding  forms  are  encoun- 
tered. Structurally  there  are  a  thick  capsule,  a  clear  one,  and  a 
central  staining  protoplasm,  which  is  granular  and  sometimes  has 
from  one  to  three  vacuoles.  The  usual  diameter  is  ten  microns. 
There  are  a  few  giant  cells  in  the  cutis,  which  are  immediately 
surrounded  by  leukocytes  and  plasma  cells,  a  tubercle-like  zone 
of  endotheloid  cells  being  absent.  As  the  tissue  was  exhausted, 
further  staining  could  not  be  done.  Nothing  can  be  said  relative 
to  the  presence  of  tubercle  bacilli. 

A  satisfactory  discussion  of  the  clinical  diagnosis  cannot  be 
given.  As  stated,  the  diagnosis  of  tuberculosis  of  the  knee-joint 
was  made.  According  to  the  tissue  findings,  it  is  probable  that  the 
chnicians  dealt  with  a  case  of  oidiomycosis  rather  than  tubercu- 
losis. 


IV.    ANALYSIS  OF  BLASTOMYCETIC  INFECTION  OF  THE  SKIN. 
CLINICAL  DATA. 

Although  the  facts  seem  to  indicate  that  the  so-called  pro- 
tozoic  disease,  Busse's  Saccharomycosis  hominis,  and  Gilchrist's 
blastomycetic  dermatitis  are  related  processes,  the  infection  as 
it  occurs  in  the  skin  obviously  claims  our  chief  attention  at  this 
time. 

In  all,  twenty-six  cases  of  so-called  "blastomycetic  dermatitis" 
have  been  collected,  the  clinical  histories  of  which  (except  Case  XI) 
have  been  given  very  briefly  here  by  the  original  observers. 

A  large  proportion  of  the  patients  have  been  laboring  men  in 
various  capacities,  and  perhaps  more  susceptible  to  infection 
because  of  the  greater  likelihood  of  traumatism.  Eight  were 
women,  all  engaged  in  housekeeping.  About  half  of  the  cases  were 
of  rather  low  station,  but  only  a  few  lived  under  conditions  where 
filth  diseases  are  ordinarily  found.  Many  had  suffered  from  the 
usual  diseases  of  childhood,  or  from  some  later  febrile  process 
(typhoid  fever,  smallpox,  rheumatism),  which  appeared  to  be  in 
no  way  associated  with  the  development  of  the  skin  disease.  It 
is  important  to  note  that,  though  always  diUgently  searched  for, 
a  history  of  syphilitic  infection,  or  of  any  of  the  usual  manifesta- 


I20  Contributions  to  Medical  Science 

tions  of  syphilis,  has  in  no  case  been  obtained;  this  includes,  in 
many  cases  at  least,  inquiry  as  to  the  condition  of  the  wife  and 
children  of  the  patient;  similarly  no  evidence  of  hereditary  syphilis 
was  discovered  in  any  case.  Two  (Hyde,  Hektoen,  and  Bevan, 
and  Busse)  might  have  been  tuberculous,  while  others  were  entirely 
free  from  any  suspicion  of  tuberculosis. 

The  family  history  has  been  uneventful  or  entirely  negative  in 
most  instances.  In  one,  however,  five  members  of  the  family 
had  died  of  tuberculosis  (Hyde,  Hektoen,  and  Bevan),  and  the 
mother  of  another  succumbed  to  the  same  disease  (F.  G.  Harris). 
There  was  a  doubtful  family  history  of  carcinoma  in  one  case.  No 
patient  knew  of  a  similar  disease  in  his  immediate  family  or  ante- 
cedents. 

When  such  a  small  number  of  individuals  is  infected  with  a 
disease,  the  specific  cause  of  which  is  probably  widespread,  we  must 
suppose  that  some  unusual  conditions  determine  the  infection; 
conditions  relating  either  to  the  patient  or  the  micro-organism  or 
perhaps  both.  It  seems  probable  that  the  oidia  are  subject  to 
great  variations  in  their  pathogenicity,  just  as  individuals  are  in 
susceptibiHty,  and  that  the  mutual  adaptabihty  requisite  for 
infection  is  not  often  realized. 

Among  the  nationalities  represented  by  the  patients  are  Ameri- 
can, German,  Irish,  and  PoHsh.  Infection  in  the  foreign-born 
occurred  in  all  cases  after  immigration  to  America.  Previous  or 
present  residence  in  America  seems  to  have  had  no  relation  to  the 
development  of  the  disease.  The  oldest  patient  was  a  German  of 
seventy-three  years  (Case  VIII),  and  the  youngest  a  little  more 
than  thirty  years;   the  average  age  at  inception  being  about  forty. 

Physical  examination  has,  almost  without  exception,  shown  the 
patients  to  be  in  excellent  general  health.  Of  the  cases  I  have 
examined  personally  none  exhibited  general  or  visceral  diseases; 
Case  VII  showed  moderate  arteriosclerosis,  incident  to  his  years. 

The  following  list  of  areas  which  have  been  attacked  in  the 
different  cases  indicates  that  probably  no  part  of  the  skin  surface 
is  immune:  scalp,  face,  including  ears,  eyelids,  nose,  cheek,  chin, 
and  lip;  neck,  chest,  back,  buttocks,  forearm,  and  arm;  scrotum, 
thigh,  and  leg;   backs  of  hand  and  foot;   9,nd  in  one  case  the  sole 


Oidiomycosis  of  the  Skin  and  Its  Fungi  121 

of  the  foot.  In  none  of  those  cases  where  the  palpebrae  have  been 
involved  has  the  conjunctiva  or  the  orbit  been  invaded,  though  the 
former  has  suffered  severely  from  exposure  and  secondary  infec- 
tions. [In  Busse's  case  of  Saccharomycosis  hominis,  however,  the 
eye  was  invaded;  the  same  has  occurred  in  the  protozoic  disease.] 
For  unknown  reasons  the  scalp  also  usually  resists  invasion. 

The  first  finding  on  the  part  of  the  patient,  in  all  cases,  is  a 
sluggish  red  papule  or  pustule.  If  there  has  been  a  preceding 
traumatism  he  may  observe  that  either  the  wound  does  not  heal 
(Case  IV)  or  that  apparent  heahng  occurs,  the  papule  or  pustule 
not  appearing  for  several  days  (Hessler),  or  weeks  later  (Stelwagon). 
The  primary  papule  ordinarily  becomes  pustular  in  a  short  time, 
and  the  apex  of  the  pustule  transformed  into  a  crust,  which  when 
removed  is  found  to  cover  an  irregular,  elevated,  reddened  base 
which  secretes  a  small  amount  of  glairy  muco-pus;  a  narrow  red 
areola  appears  early.  Extension  takes  place  steadily  so  that  in 
from  two  to  four  or  six  months  the  lesion  has  a  diameter  of  one 
to  two  inches.  In  the  meanwhile  the  rough  granular  condition 
of  the  base  becomes  greatly  exaggerated  and  gradually  a  coarse 
papillomatous  or  villiform  surface  results,  the  processes  of  which 
are  separated  by  deep,  irregular  clefts  which  are  filled  with  pus. 
The  appearance  sometimes  is  decidedly  cauliflower-like,  the 
verrucous  tissue  rising  precipitously  above  and  overhanging  the 
surrounding  skin.  In  other  cases  the  papillomatous  tissue  is  more 
flattened  and  shows  little  of  the  cauliflower  appearance;  this  is 
always  the  case  in  the  process  of  healing. 

The  skin  immediately  surrounding  the  verrucous  tissue  forms 
a  characteristic  areola.  In  the  most  extreme  condition  it  is  red 
or  bluish-red,  tumid,  rises  somewhat  to  meet  the  base  of  the  ulcer, 
possesses  an  unbroken  horny  layer,  and  is  studded  with  numerous 
sub-  or  intra-epithelial  abscesses,  which  appear  as  minute  yellowish 
points,  I  to  1.5  millimeters  in  diameter.  On  being  pricked  they 
yield  a  droplet  of  sticky,  glairy  pus  in  which  the  organisms  are 
easily  demonstrated.  The  degree  in  which  these  findings  are 
present  varies  in  different  cases  and  in  the  same  case  at  different 
times. 

When  the  lesion  has  covered  an  area  of  two  inches,  or  perhaps 


122  Contributions  to  Medical  Science 

less,  the  center  loses  in  part,  or  even  completely,  its  papillomatous 
surface,  becomes  depressed  below  the  level  of  the  surrounding 
tissue,  and  either  appears  red,  moist,  and  granulating,  or  it  may- 
be dry,  and  show  cicatricial  healing. 

In  most  cases  the  disease  exhibits  periods  of  rapid  progression 
interrupted  by  periods  of  relative  quiet.  During  rapid  extension 
the  whole  surface  is  more  swollen,  tumid,  and  red,  secretes  an 
offensive  pus  more  freely,  the  areola  becomes  broader,  more  turges- 
cent,  and  contains  a  larger  number  of  abscesses,  the  pus  of  which 
is  decidedly  bloody.  In  an  interim  of  quietude  the  surface  flattens 
out  noticeably,  is  dryer,  the  areola  narrower  and  flatter  with  fewer 
abscesses,  which  now  contain  little  or  no  blood. 

Case  VII  illustrates  strikingly  the  possibihty  of  rapid  pro- 
gression. During  the  first  two  or  three  months  that  the  patient 
was  under  observation,  the  process  remained  almost  stationary 
under  large  doses  of  iodide  of  potassium.  However,  even  while 
he  was  taking  three  hundred  grains  of  the  drug  daily,  rapid  exten- 
sion set  in  and  the  borders  extended  from  one-half  to  three-fourths 
of  an  inch  in  a  month's  time.  It  then  quieted  down  for  about  two 
weeks,  when  a  still  more  rapid  progression  developed  which  sub- 
sided only  after  continued  ingestion  of  increased  doses  of  the 
iodide.  The  periods  of  rest  and  progression  were  characterized 
by  the  features  mentioned  above. 

The  disposition  of  the  disease  to  creep  gradually  from  the 
point  of  origin  to  rather  distant  areas  is  well  illustrated  in  the 
case  of  Gilchrist  and  Stokes,  where  it  began  as  a  papule  over  the 
left  mastoid  process,  whence  it  encircled  the  left  ear,  invaded  the 
left  cheek,  chin,  palpebral,  and  supraorbital  skin,  successively, 
then  the  bridge  of  the  nose,  and  finally  similar  points  on  the 
right  side  of  the  face;  the  older  areas  cicatrizing  as  the  borders 
advanced. 

The  discomfort  attending  the  process  varies  from  a  sensation 
of  soreness  to  severe  pain,  depending  apparently  on  the  location 
of  the  lesion  and  the  degree  of  temporary  virulence  and  mixed 
infection.  More  pain  is  experienced  when  it  involves  a  dependent 
area,  or  skin  which  is  necessarily  subject  to  frequent  tension,  as 
that  over  the  dorsum  of  the  hand  (Hyde,  Hektoen,  and  Bevan) ; 


Oidiomycosis  of  the  Skin  and  Its  Fungi  123 

the  patient  of  Gilchrist  and  Stokes,  whose  face  was  chiefly  involved, 
suffered  little. 

That  a  particular  focus  may  heal  completely  with  no  treatment 
is  shown  in  the  cases  of  Gilchrist  and  Stokes,  Anthony  and  Herzog, 
Coates,  Stelwagon,  and  in  Cases  II  and  IX  of  this  series.  The 
extension  is  accomplished  by  a  direct  invasion  of  the  contiguous 
skin,  in  which  it  differs  from  the  sporotrichal  infection  described  by 
Schenck  and  by  Hektoen  and  from  the  "  blastomycetic "  disease 
in  animals  described  by  Fermi  and  Aruch,  and  by  Tokishige. 

As  stated,  heahng  begins  by  the  gradual  disappearance  of  the 
verrucous  tissue  from  the  central  or  oldest  part  of  the  lesion. 
It  is  difficult  to  know  what  becomes  of  the  papillae  in  all  cases; 
some  melt  away  obscurely,  others  remain  as  prominent  shafts 
which  become  desiccated,  horny,  or  papyraceous,  and  eventually 
fall  off,  leaving  an  absolutely  smooth  base.  Concomitant  with  or 
preceding  this,  the  number  of  surface  abscesses  becomes  smaller 
and  the  discharge  of  pus  consequently  less.  Eventually  the  result 
is  cicatricial  epidermization.  If,  however,  the  process  has  been 
so  intense  as  to  destroy  the  epidermis  over  a  considerable  area,  a 
granulating  surface  supervenes,  which  may  continue  raw,  oozing, 
and  exuberant  for  months  after  the  remainder  of  the  surface  has 
healed.  The  same  steps  in  heahng,  of  course,  occur  whether 
the  result  is  accompHshed  spontaneously  or  by  medication.  As 
healing  progresses,  the  central  cicatricial  area  becomes  larger  and 
larger,  the  peripheral  fringe  of  papillae  more  and  more  narrow  and 
flat,  and  the  surrounding  areola  gradually  disappears.  The  last 
vestiges  of  the  disease  are  usually  seen  at  the  periphery  of  the  lesion, 
in  the  form  of  a  slightly  reddened  zone  and  a  few  scattered  stumpy 
processes  which  either  are  absorbed  or  dry  up  and  desquamate. 

In  the  early  stage  of  cicatrization  it  is  not  uncommon  to  find 
scattered  miliary  abscesses,  containing  a  few  fungous  cells,  in  the 
cicatrix  itself  even  when  the  verrucous  tissue  has  largely  disap- 
peared (Cases  IV  and  VII) .  The  study  of  sections  shows  that  the 
organisms  may  remain  imbedded  in  the  deeper  granulation  tissue 
long  after  heahng  has  been  well  started  (Case  IV).  This  fact 
seems  to  explain  the  recurrence  of  the  disease  when,  supposing 
cure  had  been  established,  the  patient  ceases  medication. 


124  Contributions  to  Medical  Science 

The  resultant  scar  is  at  first  thick  and  leathery,  but  in  the 
course  of  a  few  months  or  a  year  becomes  exceedingly  thin  and 
delicate,  freely  movable  over  the  underlying  tissue,  and  having, 
in  some  instances,  a  pink  color  not  far  removed  from  that  of  normal 
skin.  The  surface,  however,  remains  somewhat  glistening,  perhaps 
slightly  wrinkled,  and  is  often  marked  by  telangiectases.  The 
line  of  demarkation  from  the  surrounding  skin  is  quite  sharp. 
Hypertrophic  scars  have  formed  in  none  of  the  cases  of  this  series, 
although  Gilchrist  noted  such  an  event. 

The  amount  of  resulting  deformity  depends  naturally  upon 
the  area  involved.  In  Case  VI  the  whole  dorsum  of  the  hand 
had  been  affected,  yet  the  newly  formed  epidermis,  although 
cicatriform,  was  perfectly  lax  and  caused  no  deformity.  In  Case 
X  also  nearly  the  whole  anterior  surface  of  the  fore-arm  had  been 
traversed,  yet  the  scar  is  now  barely  perceptible.  The  amount 
of  contraction  in  Case  IV  is  not  known,  as  the  patient  has  been  lost 
sight  of.  It  is  chiefly  in  those  cases  where  the  palpebral  tissues 
have  been  involved  that  harmful  contraction  has  occurred.  A 
high  grade  of  ectropion  results  invariably,  leading  to  more  or  less 
severe  conjunctivitis  or  keratitis,  and  even  complete  loss  of  vision  as 
in  Case  VIII;  these  conditions  being  the  result  of  exposure  and 
bacterial  infections  rather  than  invasion  by  the  oidium. 

The  "protozoic"  disease  has  most  frequently  begun  as  a  visceral 
infection  (i.e.,  in  the  lungs).  Rixford  and  Gilchrist's  first  case, 
however,  shows  that  it  may  originate  in  the  skin.  Apparently 
metastases  occur  much  more  readily  from  the  lungs  to  the  skin 
than  in  the  opposite  direction. 

Whether  the  disease  is  primarily  or  secondarily  located  in  the 
lungs  the  subsequent  course  is  very  similar  to  that  of  pulmonary 
tuberculosis,  where  abscesses  and  miliary  nodules  are  formed. 
Fever,  rising  at  night  to  ioi°-io3°  F.,  and  falling  in  the  morning, 
is  present  until  death  approaches,  when  the  temperature  may  be 
subnormal.  Prominent  symptoms  are  cough,  profuse  expectora- 
tion, pleuritic  pains,  and  the  physical  signs  common  to  pulmonary 
tuberculosis;  severe  sweating  has  also  been  noted.  Extreme 
emaciation  and  asthenia  supervene,  and  vomiting  and  stupor  may 
be  terminal  symptoms.     The  urine  shows  nothing  characteristic 


Oidiomycosis  of  the  Skin  and  Its  Fungi  125 

and  may  be  quite  normal.  After  general  or  extensive  pulmonary 
involvement  has  begun  life  may  be  prolonged  for  four  months  to 
a  year. 

Metastatic  foci  in  the  skin  may  appear  as  subcutaneous  nodules 
and  abscesses,  which  may  persist  as  such  for  some  time  before 
involving  the  epidermis;  or  the  epidermis  may  be  involved  almost 
from  the  start.  Metastatic  skin  lesions  appear  to  be  more  destruc- 
tive than  the  lesions  of  "blastomycetic  dermatitis"  and  cause 
a  freer  formation  of  pus,  in  which  the  organisms  are  said  to  be 
present  in  astounding  numbers.  Clinically  the  foci  in  the  skin 
appear  much  like  those  of  ''blastomycetic  dermatitis."  Rixford 
and  Gilchrist  describe  the  same  papillomatous  surface  and  cauli- 
flower appearance.  Some  of  the  lesions  appear  tumor-like,  how- 
ever, resembUng  the  tumor  formation  in  Mycosis  fungoides  (Wer- 
nicke, Posadas,  D.  W.  Montgomery).  The  cHnical  history, 
physical  findings,  and  the  course  of  Busse's  case  of  Saccharomy- 
cosis  hominis  are  similar  to  those  of  the  "protozoic"  disease.  In 
both  were  found  metastatic  foci  in  various  internal  organs  and  in 
certain  bones  (tibia,  ulna,  rib,  and  frontal).  It  seems,  however, 
that  the  skin  lesions  in  Busse's  case  did  not  develop  a  verrucous 
surface;  that  the  infection  was  so  virulent,  or  the  patient's  tissues 
so  weakened,  that  the  changes  were  almost  entirely  destructive. 

In  none  of  the  cases  of  so-called  blastomycetic  dermatitis,  have 
there  been  general  disturbances,  such  as  fever,  headache,  and 
nausea,  and  the  urine  has  been  persistently  normal.  Exception 
must  be  made  in  Case  V,  since,  as  stated,  it  is  now  known  that  the 
disease  eventually  became  pulmonary. 

It  appears  striking  that  out  of  twenty-six  cases  of  "blasto- 
mycetic dermatitis"  only  one  resulted  in  general  invasion  and 
death,  while  all  cases  described  as  "protozoic"  have  been  fatal. 
The  condition  would  seem  to  militate  against  the  conception  that 
the  two  processes  represent  a  disease  unit.  This  difference  in 
termination  seems  explainable,  however,  by  the  fact  that  in  the 
"protozoic"  cases  the  infection  was  usually  primary  in  the  lungs, 
where  extension  is  much  easier  than  in  the  skin,  metastases  occur 
with  greater  ease,  and  general  toxic  disturbances  are  more  easily 
produced.     In  one  of  Rixford  and  Gilchrist's  cases  of  ''protozoic" 


126  Contributions  to  Medical  Science 

infection,  where  the  primary  infection  was  in  the  skin,  many 
years  passed  before  general  invasion  occurred;  in  which  respect  it 
is  quite  comparable  to  Case  V  of  this  series.  The  observations  are 
yet  too  few  and  cover  too  short  a  time  to  be  able  to  say  in  what 
proportion  of  cases  a  primary  cutaneous  infection  would  lead  to 
general  involvement. 

diagnosis. 

The  process  in  the  skin  has  now  been  studied  clinically  and 
histologically  in  so  many  cases,  and  with  such  uniform  findings, 
that  a  characteristic  clinical  appearance  and  course,  as  well  as  a 
characteristic  histopathology,  may  be  considered  well  estabhshed. 
These  have  been  described  in  previous  pages. 

It  is  to  be  emphasized  that  the  most  ready  means  of  making 
an  immediate  diagnosis  is  to  examine  the  contents  of  the  mihary 
abscess,  or  a  fragment  of  verrucous  tissue,  microscopically,  for 
the  fungous  cells.  The  pus  may  be  mounted  either  in  a  30  per 
cent  KOH  or  NaOH  solution,  or  in  water,  and  examined  imme- 
diately. If  the  alkaline  mounts  are  allowed  to  stand  for  an  hour 
or  more,  the  field  will  be  partially  cleared  of  tissue  cells. 

Probably  the  disease  has  been  most  often  confounded  with 
verrucous  tuberculosis,  both  cHnically  and  histologically.  Case 
XI,  on  the  strength  of  microscopic  examination,  had  been  previously 
called  tuberculosis,  and  Cases  IX  and  XII  were  considered  tuber- 
culous, cHnically. 

Furthermore,  the  enormous  hyperplasia  of  the  epithelium 
suggests  carcinoma  so  strikingly  that  many  cases  doubtless  have 
been  passed  over  as  carcinoma  of  the  skin. 

In  spite  of  certain  similarities,  there  are  clinical  and  histo- 
logical features  which  separate  the  disease  from  both  tuberculosis 
and  carcinoma  of  the  skin.  It  is,  first  of  all,  probable  that  the 
current  conception  of  verrucous  tuberculosis  has  been  colored 
somewhat  by  the  inclusion  of  cases  under  that  title  which  really 
were  examples  of  oidiomycosis  of  the  skin.  Without  entering 
into  a  detailed  discussion  of  the  characteristics  of  verrucous  tuber- 
culosis it  will  be  sufficient  to  point  out  the  essential  differences 
between  the  two  diseases.     The  course  of  oidiomycosis  of  the  skin 


Oidiomycosis  of  the  Skin  and  Its  Fungi  127 

is  very  rapid  compared  to  that  of  tuberculosis,  and  in  only  two  or 
three  months  may  cause  an  ulcer  an  inch  or  more  in  diameter.  As 
extension  is  more  rapid,  so  is  the  healing  process.  In  four  to  six 
months  one  may  find  a  lesion  two  inches  in  diameter,  the  center  of 
which  has  healed  largely,  while  the  periphery  continues  to  advance. 
Such  rapid  strides  of  invasion  and  healing  are  quite  foreign  to 
verrucous  tuberculosis.  The  verrucous  processes  in  oidiomycosis 
of  the  skin  are  soft,  friable,  succulent,  and  attain  large  dimensions; 
while  in  tuberculosis  they  are  often  smaller,  drier,  hard,  and 
somewhat  horny.  The  purulent  secretion  of  the  former  exceeds 
that  usually  seen  in  the  latter.  Finally,  the  areola,  marked  by 
many  minute  abscesses,  which  is  so  characteristic  of  oidiomycosis, 
is  entirely  absent  in  verrucous  tuberculosis.  Guinea-pigs  inocu- 
lated with  oidiomycotic  skin  have  never  developed  tuberculosis; 
an  abscess  may  result,  however,  from  which  may  be  cultivated 
the  fungus  peculiar  to  the  case.  The  epithelial  hyperplasia  and 
cell  infiltrate  may  be  very  similar  in  the  two  diseases;  but  the 
amount  of  cell  infiltrate  in  oidiomycosis  greatly  exceeds  that  in 
tuberculosis.  The  microscopic  intra-epithelial  abscesses  always 
found  in  the  former  are  absent  in  the  latter.  In  addition,  the 
micro-organisms  found  in  the  two  diseases  are  characteristic  and 
differential.  The  giant  cells  and  tubercles  in  the  two  diseases  are 
similar  in  many  respects.  (See  "Special  Histological  Features.") 
In  separating  oidiomycosis  from  carcinoma  of  the  skin,  a  fact 
of  prime  importance  is  that  metastases  of  neoplastic  epithelial 
cells  do  not  occur  in  the  former;  where  metastases  have  occurred 
they  appear  to  consist  solely  of  the  fungous  cells.  The  rarity  with 
which  oidiomycosis  of  the  skin  invades  even  neighboring  lymph 
glands  has  already  been  referred  to.  Carcinomata  of  the  skin  do 
not  become  papillomatous  ordinarily;  certain  mushroom-like  or 
cauliflower-Uke  carcinomata  of  the  skin,  which  are  spoken  of,  are 
conspicuously  neoplastic,  even  clinically.  The  presence  of  miliary 
abscesses  in  the  areola  differentiates  carcinoma,  as  it  does  tubercu- 
losis, from  oidiomycosis  of  the  skin.  As  to  histology,  carcinomata 
do  not  have  histologic  miliary  abscesses,  a  budding  fungus  has 
never  been  satisfactorily  demonstrated  in  them.  Isolated  nests 
of  epithelial  cells  penetrate  the  subcutaneous,  fatty,  and  muscular 


128  Contributions  to  Medical  Science 

tissue,  and  the  amount  of  surrounding  inflammatory  cell  infiltrate 
is,  ordinarily,  moderate ;  the  contrary  conditions  prevail  in  oidiomy- 
cosis of  the  skin. 

In  regard  to  the  opinion  held  by  some,  that  the  disease  may  be 
a  form  of  syphilis,  it  seems  almost  sufficient  to  say  that  in  no 
reported  case  of  " blastomycetic  dermatitis"  has  a  history  of 
syphilis  in  any  form  been  obtained.  The  fact  that  improvement 
or  cure  follows  the  administration  of  the  iodide  of  potassium  seems 
to  be  the  only  justification  for  this  belief.  For  three  reasons  this 
position  cannot  be  held :  (i)  The  fact  that  certain  types  of  actinomy- 
cosis improve  or  heal  under  the  ingestion  of  the  iodide  destroys 
the  unconditional  specificity  of  the  drug  as  a  differential  test  for 
sj^hilis.  (2)  Oidiomycosis  demands  for  cure  a  much  larger 
dosage  and  a  longer  term  of  treatment  than  any  case  of  cutaneous 
syphilis  can  withstand.  (3)  Mercurials  are  not  effective  in 
reducing  oidiomycosis  of  the  skin. 

There  seems  to  be  no  reason  for  confounding  Kaposi's  Syphihs 
cutanea  vegetans  with  this  condition.  The  histology  of  syphilis 
is  so  clearly  different  that  detailed  comparisons  seem  unnecessary. 

The  diagnosis  of  the  condition  of  the  lungs  may  be  dismissed 
with  the  statement  that  its  marked  similarity  to  pulmonary 
tuberculosis  has  given  rise  to  confusion.  It  seems  probable  that 
further  observation  will  show  that  a  clinical  differentiation  can  be 
made  by  microscopic  examination  of  the  sputum. 

prognosis. 

That  the  disease,  when  primarily  in  the  skin,  is  not  often  of 
great  malignancy,  is  indicated  by  its  tendency  to  remain  localized 
even  for  ten  or  more  years.  It  is  not  certain,  in  any  case,  that 
generalization  ever  would  result.  However,  Rixford  and  Gil- 
christ's first  case  of  the  "protozoic"  disease  and  Case  V  of  this 
series  furnish  convincing  proof  that  a  fatal  generahzation  may 
occur.  When  confined  to  the  skin,  the  disease  yields  readily  to 
proper  treatment,  with  a  varying  amount  of  cicatricial  deformity. 
Spontaneous  healing  is  possible,  at  least  in  part.  In  several  cases 
where  there  were  multiple  foci,  certain  of  the  latter  healed  entirely 
with  no  treatment.     As  stated,  in  progressive  lesions,  spontaneous 


Oidiomycosis  of  the  Skin  and  Its  Fungi  129 

healing  occurs  in  the  oldest  parts  as  the  border  advances.  Although 
it  has  not  been  observed,  it  seems  probable  that  the  disease  may 
at  times  make  an  absolute  spontaneous  recovery. 

TREATMENT. 

The  observation  made  by  Professor  A.  D.  Bevan  that  the 
disease  improved  under  the  internal  administration  of  the  iodide 
of  potassium  has  been  substantiated  by  the  experience  of  others, 
particularly  Professors  Hyde  and  Montgomery.  Three  cases 
have  recovered  completely,  and  four  more  have  improved  greatly 
under  this  treatment,  combined  with  the  apphcation  of  antiseptics 
to  the  ulcers.  It  is,  as  usual,  advisable  to  begin  with  about  ten 
grains  three  times  a  day,  and  increase  the  dose  steadily.  Improve- 
ment may  begin  when  twenty  grains  is  reached;  this  improvement 
usually  has  been  found  to  be  only  temporary,  however,  making  it 
necessary  to  increase  the  dosage  to  fifty,  seventy-five,  one  hundred, 
or  even  to  one  hundred  and  fifty  grains  three  times  daily.  In  the 
treatment  of  Case  VII  Httle  improvement  resulted  until  one 
hundred  grains  was  reached,  then  healing  progressed  rapidly  for 
a  time;  it  again  became  stationary  and  it  was  necessary  to  go 
first  to  one  hundred  and  twenty,  then  to  one  hundred  and  fifty 
grains  per  dose,  when  rapid  improvement  again  set  in. 

Locally  the  use  of  hot  antiseptics  is  of  great  value  in  disposing 
of  secondary  infection  and  reheving  the  soreness.  Hot  boracic  acid 
and  corrosive  sublimate  solutions  have  been  used  principally. 
It  was  thought  that  the  latter,  when  applied  continuously,  would 
penetrate  the  tissues  sufficiently  to  check  the  infection;  this  result, 
however,  was  not  realized  in  the  treatment  of  Case  VII,  and  the 
boracic  acid  applications  were  resumed.  Also  no  good  effect  was 
noted  from  the  use  of  the  tincture  of  iodine  over  the  surface,  or 
the  subcutaneous  infiltration  of  an  iodo-potassic-iodide  solution. 
No  marked  improvement  has  occurred  in  any  case  under  merely 
local  antiseptic  treatment. 

Total  excision  of  limited  areas  has  given  perfect  results  (Cases 
I  and  IX).  This  is  easily  possible,  owing  to  the  superficial  location 
of  the  infection.  Thorough  curetting  followed  by  cauterization 
would  seem  to  offer  good  results,  a  general  anesthetic,  of  course, 


I^O 


Contributions  to  Medical  Science 


being  necessary.  When  cicatricial  ectropion  of  the  eyelids  has 
resulted,  care  of  the  eye  is  quite  necessary  to  prevent  conjunctivitis 
and  keratitis.  Dr.  Dodds,  of  the  Illinois  Eye  and  Ear  Infirmary, 
has  corrected  the  marked  ectropion  resulting  in  Case  II  by  plastic 
operations. 

The  continuous  administration  of  the  iodide  of  potassium,  then, 
in  large  doses,  offers  the  best  hope  of  cure,  where  excision  cannot 
be  practiced.  The  necessary  duration  of  treatment  varies  from 
three  to  four  months  to  a  year  or  perhaps  longer.  With  this, 
however,  the  use  of  local  antiseptics  should  be  combined. 

pathogenesis. 

It  has  been  stated  that  no  part  of  the  skin  surface  is  proof 
against  invasion.  The  scalp,  palmar,  and  plantar  tissues,  however, 
seem  provided  with  considerable  resistance  to  the  infection,  and 
the  mucous  surfaces,  in  particular,  usually  escape.  (The  transition 
surface  of  the  lower  Hp,  in  Case  I,  was  invaded.) 

From  the  statements  made  in  literature  it  may  be  gathered, 
also,  that  many  of  the  internal  organs  are  equally  susceptible, 
when  an  internal  focus  is  once  estabhshed,  as  in  cases  of  the  "pro- 
tozoic"  disease,  Busse's  Saccharomycosis  hominis,  and  Case  V 
of  this  series. 

The  indications  are,  as  previously  suggested,  that  the  original 
point  of  infection  may  lie  either  in  the  skin  or  in  one  of  the  internal 
organs,  particularly  the  lungs.  In  the  condition  described  as 
blastomycetic  dermatitis  it  seems  clear  in  most  cases  that  the  skin 
was  the  part  primarily  invaded.  In  several  cases  of  the  *' pro  to- 
zoic"  disease  it  seems  equally  clear  that  the  lungs  were  first  invaded. 
Busse  and  Buschke  do  not  agree  concerning  the  infection  atrium 
in  the  former's  case,  Busse  considering  it  visceral,  and  Buschke 
that  it  was  first  situated  in  the  skin. 

No  sweeping  statement  can  be  made  concerning  the  mode  of 
primary  invasion  of  the  skin.  Quite  surely,  however,  three  of 
the  infections  followed  traumatisms;  in  one  case  a  razor-cut 
(Hessler),  in  the  second  a  scratch  or  bite  by  a  cat  (Stelwagon), 
and  in  the  third  an  abrasion  made  by  the  handle  of  a  wooden 
truck  (Case  IV).     It  cannot,  of  course,  be  known  whether  the 


Oidiomycosis  of  the  Skin  and  Its  Fungi  131 

organisms  had  previously  dwelt  on  the  intact  skin,  whether  they 
were  carried  there  by  the  instrument  of  traumatism,  or  whether 
they  were  transferred  to  the  wound  after  it  was  made.  In  Case 
I  it  seems  probable  that  there  was  a  pre-existing  abrasion  of  the 
lower  lip,  and  that  the  infection  was  derived  from  blighted  cereal 
grains,  with  which  the  patient  was  frequently  in  intimate  contact. 
How  the  organisms  penetrate  the  skin  in  the  absence  of  traumatism 
is  not  clear.  They  may  have  been  placed  superficially  in  the 
horny  layer,  or  deposited  in  a  hair  follicle  or  gland  duct ;  in  such  a 
position  the  toxins  secreted  by  the  organisms  would  perhaps  be 
sufficient  to  cause  small  areas  of  necrosis,  into  and  through  which 
the  organism  and  its  spores  might  extend.  That  is  to  say,  an 
organism  may,  itself,  be  able  to  cause  all  the  traumatism  necessary 
for  primary  invasion.  Unquestionably  the  disease,  when  once 
established  in  the  skin,  is  easily  transplanted  to  other  portions; 
this  is  illustrated  in  the  cases  of  Gilchrist  and  Stokes,  Hyde  and 
Hektoen,  Anthony  and  Herzog,  Stelwagon,  and  by  Cases  II  and 
VII  of  this  series. 

In  the  case  described  by  Coates  the  patient's  condition  pre- 
ceding the  development  of  the  foci  in  the  skin  affords  some  ground 
for  the  behef  that  the  disease  was  primarily  localized  in  the  chest, 
and  that  the  numerous  points  which  suddenly  appeared  in  the 
skin  were  of  metastatic  origin.  As  the  visceral  ailment  finally 
healed,  the  conclusion  would  naturally  be  that  it  healed  after 
causing  cutaneous  metastases.  Although  this  may  not  be  possible, 
it  seems  unlikely,  in  view  of  the  fate  of  cases  which  are  known  to 
have  had  internal  foci. 

The  three  lesions  which  originally  developed  in  Case  IX  appeared 
"at  the  same  time."  It  seems  most  probable,  of  course,  that  the 
patient  came  into  external  contact  with  the  infection  and  that 
the  three  implantations  occurred  "at  the  same  time." 

How  the  organisms  migrate  in  the  epidermis  and  corium  is  not 
clear.  There  is  no  evidence  that  they  are  motile,  and  the  adult 
cell  is  not  found  in  wandering  tissue  cells.  Still  they  are  often 
seen  isolated  in  the  epidermis  and  corium.  As  stated  elsewhere, 
it  may  be  that  minute  spores  are  produced  which  can  easily  be 
carried  in  wandering  cells,  or  in  the  lymph  currents. 


132  Contributions  to  Medical  Science 

pathological  anatomy. 

As  the  gross  pathology  of  the  lesions  in  the  skin  involvement 
has  been  given  in  detail  in  the  descriptions  of  the  various  cases 
and  in  preceding  pages,  it  will  sufl&ce  here  to  enumerate  categori- 
cally the  principal  points. 

Primary  lesion. — A  reddish  papule  which  is  soon  transformed 
into  a  crusted  pustule. 

Intermediate  appearance. — A  prominently  elevated  ulcer,  pos- 
sessing a  granular  base,  secreting  glairy  muco-pus,  which  dries 
to  form  crusts. 

Fully  developed  lesion. — An  elevated,  coarsely  papillomatous, 
sharply  circumscribed  ulcer,  which  secretes  pus  freely,  contains 
many  minute  abscesses,  and  which,  cauhflower-like,  may  overhang 
the  surrounding  reddened  areola,  which  also  contains  numerous 
miHary  abscesses.  Later,  the  central  portion  is  depressed,  and 
almost  entirely  free  from  papillae  and  abscesses,  concomitant 
with  heahng  and  cicatrization. 

Cross-section. — Two  zones  are  visible,  a  papillary  and  a  deeper 
homogeneous  zone  containing  minute  macroscopic  abscesses  and 
small  hemorrhages.  The  papillary  zone  is  made  up  of  two  portions, 
the  deep,  which  is  a  palisade-Hke  layer  of  closely  united  papillae,  and 
the  superficial,  composed  of  a  smaller  number  of  papillae,  which 
rise  above  the  general  level  of  the  ulcer  and  are  separated  by  clefts. 

The  study  of  sections  shows  the  following  points  in  relation 
to  the  gross  appearances:  That  portion  of  the  surface  pus  which 
adheres  to  the  tissue  contains  many  polymorphonuclear,  a  few 
mononuclear  leukocytes,  few  or  no  eosinophiles,  no  plasma-  or 
mast-cells,  masses  and  layers  of  desquamated  horny  material, 
many  bacteria,  and  very  few  of  the  specific  fungous  cells. 

The  papillae  are  shafts  of  the  rete  mucosum,  which  are  sheathed 
by  a  thin,  imperfectly  cornified  layer,  and  each  is  suppUed  with 
a  central  vascularized  cone  of  mesoblastic  tissue.  Each  of  the 
processes  constituting  the  deeper  portion  of  the  papillary  zone  is 
closely  cemented  to  those  contiguous  by  septa  of  horny  material. 
The  deep  surface  of  the  epidermis  is  very  irregular  because  of  the 
processes  which  penetrate  the  homogeneous  zone.  The  homo- 
geneous zone  is  largely  a  mass  of  infiltrating  leukocytes,   new 


Oidiomycosis  of  the  Skin  and  Its  Fungi  133 

fixed  tissue  cells,  minute  abscesses  and  vessels.  It  has  supplanted 
the  corium  proper,  and  the  superficial  portions  of  the  subcutaneous 
tissue,  which  have  lost  all  or  nearly  all  their  elastic  fibers;  the 
remaining  collagenous  tissue  is  very  edematous.  The  line  of 
demarkation  from  the  underlying  fatty  tissue  is  quite  sharp. 

The  miliary  abscesses  are  located  chiefly  in  the  deeper  epitheHal 
tissue,  and  in  the  homogeneous  zone.  Many  smaller  ones,  however, 
are  found  in  the  surface  papillae. 

The  fungous  cells  are  distributed  numerically  as  follows:  (i)  the 
largest  number  is  found  in  the  abscesses  of  the  corium  and  deep 
epithelial  tissue;  (2)  moderate  numbers  are  found  in  more  super- 
ficial epidermal  abscesses;  (3)  a  few  are  encountered  between  the 
cells  of  the  rete  mucosum,  and  in  the  granulation  tissue  of  the 
corium,  unassociated  with  large  accumulations  of  leukocytes. 

As  stated  previously,  the  areolar  abscesses  contain  ordinarily 
a  droplet  of  yellowish  viscid  pus,  which,  however,  during  rapid 
progression  of  the  disease  is  strongly  tinged  with  blood.  It  is 
here  that  the  organisms  can  be  most  easily  demonstrated,  while  in 
the  surface  pus  they  are  difficult  to  find.  [On  the  contrary,  the 
organisms  were  found  in  large  numbers  in  the  surface  secretions 
in  both  the  "protozoic"  disease,  and  Busse's  case  of  Saccharomy- 
cosis  hominis.] 

It  has  been  noted  that  the  central  portion  of  the  lesion  may 
heal  readily  and  permanently,  while  the  periphery  is  still  quite 
active;  and  that  the  scar  which  eventually  forms  is  thin,  pliable, 
little  or  not  at  all  adherent,  and  may  have  almost  the  appearance 
of  normal  skin.  The  perfect  epidermization,  no  doubt,  depends 
on  the  fact  that  the  surface  at  no  time  has  lost  its  epithelial  cover- 
ing entirely,  the  latter  being  only  stimulated  to  an  abnormal  and 
excessive  proliferation.  Consequently  when  subsidence  occurs, 
a  massive  amount  of  epidermis  remains  to  be  utilized.  In  those 
few  cases,  however,  where  the  epidermis  has  been  entirely  destroyed 
over  small  areas,  the  result  is  seen  in  exuberant,  granulating  sores, 
which  may  not  heal  even  before  all  traces  of  the  active  disease  have 
disappeared.  Sections  show  that  the  active  disease  involves 
only  a  moderate  degree  of  necrosis;  that  the  changes  are  superficial, 
the  cell  infiltrate  not  entering  the  subcutaneous  tissue  to  any 


134  Contributions  to  Medical  Science 

degree;  and  that  the  elevation  and  tumor-like  appearance  of  the 
lesion  is  due  more  to  an  enormous  cell  infiltrate,  to  edema  and 
h>^eremia,  rather  than  to  excessive  reproduction  of  fixed  tissue 
elements.  These  conditions  would  seem  to  account  for  the  mild 
nature  of  the  scar  in  comparison  with  the  appearance  of  the  earlier 
lesions. 

From  what  is  given  in  literature  concerning  the  protozoic 
infection,  there  appears  to  be  no  essential  difference  between  the 
anatomy  of  its  cutaneous  lesions  and  that  of  so-called  blastomy- 
cetic  dermatitis.  But  in  Busse's  case  of  Saccharomycosis  hominis 
the  process  in  the  skin  appeared  more  destructive  than  in  any  of 
the  cases  of  this  series. 

The  changes  produced  in  animal  tissues  by  inoculation  of  the 
organisms  depend  on  the  particular  organism  and  the  animal 
employed,  and  the  character  of  the  inoculation.  The  reader  is 
referred  for  details  to  the  abstracts  of  the  cases  in  literature  and  to 
the  descriptions  of  the  cases  of  this  series.  The  different  organisms 
vary  greatly  in  virulence.  Subcutaneous  inoculations  of  pure 
cultures  commonly  cause  abscesses  in  guinea-pigs  and  rabbits,  and 
may  be  fatal  to  mice.  Intraperitoneal  injections  are  relatively 
harmless.  When  placed  intravenously,  transient  septicemia  or 
fatal  pulmonary  mycosis  may  result.  The  pathogenicity  of  the 
various  organisms  is  indicated  roughly  in  the  table  (pp.  148  f.); 
the  one  from  Busse's  case  was  highly  virulent  to  animals. 

The  subcutaneous  implantation  of  infected  human  tissue  into 
animals  may  produce  abscesses  from  which  the  organism  pecuhar 
to  the  case  may  be  cultivated,  but  tuberculosis  never  has  followed 
such  an  inoculation.  It  seems  probable  that  artificial  immunity 
is  not  readily  produced  in  animals. 

SERUM   reactions. 

Professor  Hektoen  has  found  that  the  undiluted  serum  of  a 
dog  which  had  received  successive  inoculations  of  the  organism 
from  Case  I  causes  gradual  clumping  of  the  organism  diffused  in 
bouillon.  Several  hours  elapse  before  the  fullest  extent  of  clump- 
ing possible  is  reached.  Organisms  from  other  cases  show  only 
a  sHght  degree  of  clumping  with  the  same  serum.     All  organisms 


Oidiomycosis  of  the  Skin  and  Its  Fungi  135 

grew  well  in  the  serum  and  the  production  of  mycelium  was  espe- 
cially noticeable.  Normal  dog's  serum  causes  no  clumping  of  any 
of  the  organisms;   abundant  mycelium  is  produced  in  all  cases. 

The  organism  from  the  case  reported  by  Hyde,  Hektoen,  and 
Bevan  was  repeatedly  inoculated  into  the  abdominal  cavity  of 
a  rabbit.  It  was  found  that  the  animal's  serum  would  cause 
fairly  distinct  agglutination  of  the  organism  inoculated.  Further 
studies  are  indicated  in  regard  to  immunization,  agglutination, 
and  allied  problems. 

SPECIAL  histological  FEATURES. 

Cornification. — Abnormal  or  incomplete  comification  is  found 
in  the  horny  and  granular  layer,  in  the  depth  of  the  rete,  in  single 
cells  or  groups  of  cells,  surrounding  abscesses  and  giant  cells,  and 
in  the  desquamated  rete  cells  in  the  intra-epithelial  abscesses. 
On  the  surface  this  is  manifested  by  the  retention  of  cell  nuclei 
and  keratohyalin  and  the  lack  of  fibrillation,  or  by  the  entire 
absence  of  keratohyalin  in  the  cornification  process.  The  proto- 
plasm of  such  cells  is  moderately  acidophilic.  Occasionally  a 
single  cell  is  seen  in  the  depth  of  the  rete  with  a  shrunken  nucleus 
and  strongly  acidophilic  protoplasm  and  a  few  keratohyahn 
granules.  Such  a  cell  may  be  surrounded  by  other  cells,  which 
are  more  or  less  flattened  and  may  or  may  not  contain  kerato- 
hyalin, but  are  strongly  acidophilic;  or  a  single  cell  may  contain 
another  epitheUal  cell  and  one  or  both  be  partly  cornified.  Typical 
comified  whorls  occur  with  or  without  the  intervention  of  ker- 
atohyalin; a  mass  of  leukocytes,  an  organism,  or  a  giant  cell  is 
commonly  found  in  the  center  of  a  whorl.  The  epithelial  cells 
constituting  the  wall  of  an  abscess  may  or  may  not  be  cornified, 
and  if  cornified  with  or  without  keratohyahn  formation.  In  case 
cornification  occurs  the  cells  are  flattened,  somewhat  fibrillated, 
and  take  acid  stains  deeply.  Surrounding  centers  of  irritation 
such  as  accumulations  of  leukocytes,  organisms,  or  giant  cells,  the 
prickles  are  prone  to  disappear,  giving  place  to  a  dense,  acido- 
philic, homogeneous  fiber.  Remote  from  centers  of  irritation  the 
prickles  are  present  quite  constantly.  In  the  intra-epithelial 
abscesses  are  numerous  desquamated  epithelial  cells,  which  show 


136  Contributions  to  Medical  Science 

atypical  cornification.  Some  are  perfectly  spherical,  but  appear 
dense,  take  up  eosin  strongly,  and  have  a  shrunken  nucleus;  such 
cells  may  contain  other  epithelial  cells  or  leukocytes.  The  epithe- 
lial cells  within  abscesses  often  become  fibrillated,  some  with  needle- 
shaped  nuclei,  and  others  in  which  nuclei  are  no  longer  discernible. 

Atj^ical  cornification  in  blastomycetic  dermatitis  appears  to 
be  due  to  an  abnormal  stimulation  to  growth,  brought  about  by 
the  presence  of  foreign  and  toxic  elements;  such  elements  appear 
to  be  leukocytes,  giant  cells,  and  the  parasites.  The  effect  is  a 
hastened  and  imperfect  maturation  of  the  surrounding  epithehal 
cells,  partial  or  complete  cornification,  with  or  without  the  inter- 
vention of  keratohyahn.  As  intimated  above,  this  stimulation 
may  result  in  epithehal  giant  cell  formation  instead  of  cornifica- 
tion. 

In  certain  fields  the  granoplasm  of  epithelial  cells  may  be  cen- 
tered in  a  particular  portion  of  the  cell.  This  results  in  the  so-called 
''cobblestone"  appearance. 

Epithelial  giant  cell  formation. — The  occurrence  of  numerous 
giant  cells  in  intra-epithelial  miUary  abscesses  suggested  to  Wells 
that  they  might  be  formed  from  epithelial  cells.  The  tissue  in 
certain  cases  of  blastomycetic  dermatitis  shows  numerous  giant 
cells  in  the  depth  of  the  rete,  which  may  or  may  not  be  surrounded 
by  a  few  wandering  cells.  In  one  case  where  such  giant  cells  are 
numerous  their  origin  from  epithelial  cells  seems  clear.  The 
first  step  consists  in  the  presence  of  some  foreign  element  such  as 
leukocytes  or  of  one  or  more  organisms.  Following  this,  the 
nuclei  of  adjacent  epithehal  cells  become  smaller  and  stain  more 
densely,  the  prickles  disappear,  the  cell  body  becomes  smaller 
and  fuses  with  that  of  neighboring  cells,  constituting  a  multi- 
nuclear  giant  cell  with  peripheral  disposition  of  the  nuclei.  Al- 
though mitoses  may  occur  in  this  process,  it  has  not  been  possible 
to  find  them.  A  further  accumulation  of  leukocytes  leads  gradu- 
ally to  the  formation  of  a  small  abscess;  and  so  it  happens  that 
in  most  intra-epithelial  abscesses  there  are  one  or  more  giant 
cells  possessing  epithelioid  nuclei.  These  giant  cells  contain  one 
or  more  leukocytes  or  organisms,  or  both. 

Phagocytic  epithelial  cells. — ^A  conspicuous  feature  is  the  number 


Oidiomycosis  of  the  Skin  and  Its  Fungi  137 

and  the  character  of  epithelial  cells,  which  enclose  either  leukocytes 
or  other  epithelial  cells.  A  single  rete  cell,  which  is  normal  in 
every  other  respect,  retaining  its  prickles  and  customary  staining 
properties,  may  present  a  nucleus  which  is  retracted  at  one  point, 
leaving  a  space  which  is  occupied  by  an  invading  leukocyte.  It 
is  in  the  abscesses,  however,  that  cell  invasion  or  phagocytosis 
occurs  to  a  pronounced  degree.  Case  I  shows  many  small  intra- 
epithelial abscesses,  which  contain  from  ten  to  thirty  or  forty 
such  cells  in  a  single  section.  Presumably  the  condition  begins 
by  an  epithelial  cell  taking  up  or  being  invaded  by  a  leukocyte; 
this  occurs  either  while  the  former  constitutes  a  part  of  the  abscess 
wall  or  after  its  desquamation.  At  this  point  the  cell  may  be  partly 
cornified;  and  if  partial  cornification  occurs,  further  phagocytosis 
or  cell  invasion  is  precluded.  In  the  event  that  cornification  does 
not  take  place,  the  invasion  continues  until  the  cell  is  filled  with 
from  three  to  six  or  eight  leukocytes,  the  protoplasm  forming  a 
thin  shell  and  retaining  its  nucleus  in  a  flattened  condition.  The 
protoplasm  of  the  epithelial  cell  continues  granular  until  the 
distension  is  extreme,  when  it  exists  as  a  transparent  hyalin  film 
around  the  enclosed  cells.  At  any  time  in  this  process  another 
epithelial  cell  may  be  enclosed,  and  this  cell  in  its  turn  may  take 
up  leukocytes.  It  has  been  somewhat  difficult  to  determine 
whether  this  was  purely  cell  invasion  or  purely  phagocytosis,  or 
whether  both  processes  took  place.  In  the  struggle  for  existence 
between  the  including  cell  and  the  enclosed  cells  the  former  often 
loses  its  vitality  and  further  infiltration  occurs.  It  is  not  known 
positively  whether  or  not  the  leukocytes  multiply  after  their 
envelopment.  Many  forms  of  degenerating  and  phagocytic 
epithelial  cells  appear  to  be  identical  with  certain  so-called  para- 
sites of  cancer,  described  by  Piimmer  and  others. 

Desquamated  epithelial  cells  may  undergo  granular  degen- 
eration with  virtually  no  change  of  form. 

Occasionally  one  finds  an  area  of  from  two  to  four  or  five  epithe- 
lial cells  which  have  undergone  coagulation  necrosis.  There  may 
be  no  surrounding  round  cell  infiltration. 

The  cutis. — The  dense  infiltration  of  the  cutis  is  emphasized  in 
all  cases.     Polymorphonuclear  and  mononuclear  leukocytes,  eosino- 


138  Contributions  to  Medical  Science 

philes,  plasma-  and  mast-cells  participate  in  this  infiltration. 
There  is  in  addition  a  vast  increase  of  the  fixed  tissue  element, 
including  blood  and  lymph  channels.  Polymorphonuclear  leu- 
kocytes are  often  concentrated  to  form  subepithehal  abscesses. 
Mononuclear  leukocytes  occur  in  connection  with  the  polymor- 
phonuclears, but,  together  with  the  plasma  cells,  avoid  the  areas 
of  most  intense  reaction.  Eosinophiles  occur  alike  in  remote 
areas  or  abscesses,  and  may  wander  into  the  epithehum.  Mast-cells 
of  different  types  occur  near  blood-vessels  and  gland  structure 
especially,  but  have,  moreover,  a  somewhat  general  distribution. 
They  are  also  absent  from  areas  of  intense  reaction. 

Eosinophiles. — In  a  certain  type  of  cases,  polymorphonuclear 
leukocytes  are  present  in  overwhelming  numbers,  and  it  is  noted 
also  that  in  the  same  class  of  cases  eosinophiles  are  present  con- 
stantly in  large  numbers.  These  cases  are  distinguished  chnically 
by  the  presence  of  many  visible  miliary  abscesses  and  by  the 
excessively  verrucous  appearance  of  the  diseased  surface.  Such 
cases  have  a  correspondingly  smaller  number  of  plasma  cells. 
A  second  type  of  the  disease,  which  is  characterized  by  its  some- 
what milder  clinical  appearance,  possesses  fewer  polymorphonu- 
clear leukocytes,  and  few  eosinophiles.  It  is  of  great  interest 
to  note  in  this  connection  that  in  those  cases  where  eosinophiles 
and  polymorphonuclear  leukocytes  are  present  in  such  excessive 
numbers,  the  organisms  so  far  isolated  have  been  of  a  mould- 
fungus  type.  Three  of  the  cases  and  the  one  described  by  Harris 
are  of  the  extremely  verrucous  type  and  presented  many  miliary 
abscesses  in  the  surrounding  areola.  From  two  of  our  cases  a 
mould-fungus  was  cultivated.  Culture  experiments  from  another 
failed.  In  Harris'  case  no  cultures  were  attempted.  In  the  case, 
however,  where  I  failed  to  obtain  satisfactory  cultures,  there 
developed  on  two  occasions  an  organism  which  in  its  early  condi- 
tion resembled  the  two  mould-fungi  isolated  from  similar  cases. 
It  seems  quite  possible,  then,  that  the  presence  of  such  a  large 
number  of  eosinophiles  may  be  associated  with  a  special  clinical 
type,  and  possibly  with  the  presence  of  the  mould-fungus  type  of 
organism.  At  any  rate,  there  is  clearly  an  eosinophilous  form  of 
the  disease,  histologically. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  139 

Plasma  cells  and  fibrous  tissue. — It  has  been  emphasized  in  the 
descriptions  of  the  histopathology  in  these  cases  that  the  plasma 
cell  occurs  chiefly  in  the  subacute  areas  of  the  inflammatory  process, 
particularly  in  the  peripheral  portion^  of  the  lesion,  where  new 
fibrous  tissue  is  forming  rapidly  and  in  large  quantities.  The 
fact  that  plasma  cells  are  abundant  in  the  neighborhood  of  such 
tissue  suggests  a  possible  relationship  between  the  two.  In 
studying  this  relationship,  it  has  been  of  service  to  use  a  water- 
blue  and  carbol-fuchsin  stain.  Plasma  cells  are  not  stained 
specifically  by  this  method,  but  the  chromatin  takes  up  the  fuchsin 
somewhat  specifically,  and  the  protoplasm  stains  a  sky-blue  with 
the  water-blue,  leaving  certain  unstained  spaces,  which  doubtless 
represent  the  protoplasmic  granules  of  the  cell.  In  addition,  the 
so-called  hyalin  bodies  are  brought  out  well  by  the  fuchsin,  and 
the  finest  connective-tissue  fibrillae  by  the  water-blue.  The 
sections  were  stained  in  a  j  to  ^  of  i  per  cent  aqueous  solution 
of  water-blue  for  twenty  seconds,  treated  with  water,  then  in 
Ziehl's  carbol-fuchsin  for  ten  or  fifteen  minutes,  decolorized  in 
alcohol,  to  which  had  been  added  a  small  amount  of  the  tincture  of 
iodine,  and  then  passed  through  absolute  alcohol  and  cedar  oil 
into  balsam.  The  polychrome  methylene  blue  has  been  used  as 
a  supplementary  and  corroborative  stain. 

It  is  to  be  noted  in  the  first  place  that  there  are  relatively 
few  plasma  cells  visible  in  blood-vessels.  They  exist  in  fairly 
large  numbers  immediately  surrounding  the  vessels,  and  occasion- 
ally one  is  seen  in  the  process  of  exit  from  a  vessel.  The  occurrence 
of  the  cells  immediately  surrounding  blood-vessels  is  responsible 
for  a  rough,  columnar  appearance  of  plasma  cell  groups  under  a 
low  power.  As  they  exist  here,  there  is  virtually  no  intercellular 
substance.  In  a  field  of  plasma  cells  more  remote  from  blood- 
vessels, where  the  cells  are  supposedly  older,  it  is  usual  to  see  a 
columnar  arrangement  of  a  different  sort.  There  are  straight  or 
tortuous  rows  of  cells  end  to  end,  the  columns  being  separated  by 
connective-tissue  fibers,  giving  off  delicate  branches,  which  pass 
between  and  surround  the  plasma  cells.  In  the  intercolumnar 
fibrous  tissue  are  found  occasional  elongated  cylindrical  or  oval 
nuclei,  characteristic  of  new  connective- tissue  cells.     They  have 


I40  Contributions  to  Medical  Science 

a  small  amount  of  chromatin,  the  arrangement  of  which  is  in  no 
way  suggestive  of  the  plasma  cell  nucleus.  There  is  a  distinct 
nuclear  membrane,  and  the  nucleus  itself  may  shrink  from  the 
fiber-Hke  cell  body,  forming  a  clear  perinuclear  space.  A  strictly 
columnar  arrangement  of  plasma  cells  is  not  at  all  necessary  for 
this  relationship  to  fibrous  tissue.  Groups  of  cells  everywhere 
show  the  same  conditions  except  where  they  are  newly  formed. 
Concomitant  changes  occur  within  the  plasma  cells  themselves. 
Polychrome  methylene  blue  gives  a  granular  protoplasm,  which 
stains  deeply,  and  an  eccentric  nucleus,  which  possesses  about 
eight  chromophilic  masses,  arranged  peripherally  and  connected 
by  means  of  dehcate  fibers.  Among  the  older  cells  this  type  is 
departed  from.  Those  having  the  most  intimate  relation  to  new- 
forming  fibrous  tissue  gradually  lose  their  protoplasmic  granules 
and  the  volume  of  protoplasm  decreases.  The  extreme  change 
is  seen  in  a  cell  whose  protoplasm  is  clear  or  finely  granular,  almost 
entirely  unstained,  and  possessing  an  eccentric  nucleus  character- 
istic of  the  plasma  cell.  The  cell  body  disappears  by  a  gradual 
peripheral  disintegration,  and  the  outline  becomes  uneven  and 
ill-defined.  One  often  sees  a  plasma  cell  nucleus,  with  the  merest 
fringe  of  protoplasm,  exhibiting  no  afiinity  for  the  alkaline  methyl- 
ene blue.  It  is  interesting  to  note  that  where  the  plasma  cells 
are  relatively  intact,  the  surrounding  fibrous  tissue  is  correspond- 
ingly young;  and  where  the  plasma  cells  show  the  disintegration 
spoken  of,  the  fibrous  tissue  is  more  adult  in  character.  A  close 
study  has  been  made  with  a  view  of  determining  the  transition  of 
the  plasma  cells  into  fibroblasts,  as  suggested  by  H.  F.  Harris 
and  others,  but  no  facts  have  been  found  to  substantiate  this 
theory.  There  is  always  a  clear  difference  between  the  most 
changed  plasma  cell  and  the  nuclei  of  surrounding  connective 
tissue;  that  is  to  say,  no  transition  forms  have  been  found.  That, 
however,  the  plasma  cell  is  sacrificed  in  an  indirect  way  for  the 
benefit  of  new-forming  scar  tissue  seems  very  probable. 

Hyalin  bodies  in  plasma  cells. — A  change  of  a  different  character 
is  seen  quite  constantly  in  large  numbers  of  plasma  cells,  namely, 
the  so-called  hyahn  degeneration.  In  unstained  sections  this 
substance  is  seen  as  glassy,  highly  refractive  spherules,  varying 


Oidiomycosis  of  the  Skin  and  Its  Fungi  141 

in  size  from  a  small  granule  to  one-third  or  one-fourth  of  the 
volume  of  the  cell  body.  They  are  resistant  to  strong  acids  and 
alkalies.  They  stain  well  by  Gram's  method  or  acid  fuchsin,  or 
in  Van  Gieson's  mixture,  carbol-fuchsin,  and  certain  of  them  in 
alkaline  or  polychrome  methylene  blue.  All,  however,  have 
stronger  acidophilic  than  basophilic  properties.  In  carbol-fuchsin 
preparations  the  staining  affinities  are  not  uniform.  The  small 
bodies  commonly  stain  a  deep  red,  while  the  larger  ones  have  a 
lighter  but  more  brilHant  color.  Occasionally  all  those  in  a  cell 
have  taken  up  so  little  fuchsin  that  they  appear  pale  yellow.  The 
affinities  are  equally  inconstant  for  the  other  dyes  mentioned. 
These  bodies  are  found  customarily  in  intracellular  groups.  They 
also  occur  in  groups  in  lymph  spaces  and  channels  and  between 
connective-tissue  cells.  Cells  containing  such  bodies  are  altered 
in  shape  to  accommodate  the  contents,  and  in  properly  prepared 
specimens  a  cell  nucleus  is  usually  demonstrable,  which  almost 
invariably  possesses  the  characteristics  of  the  plasma  cell  nucleus. 
Where  there  are  only  a  few  hyalin  spherules  in  the  cell,  the  proto- 
plasm stains  characteristically,  but  never  deeply,  with  polychrome 
methylene  blue.  As  they  increase  in  number,  the  basophilic 
granules  of  the  cell  body  decrease.  This  suggests  that  the  grano- 
plasm  may  be  transformed  into  these  so-called  hyalin  bodies. 
Eventually  the  cell  is  consumed  or  broken  through,  and  the  hyalin 
bodies  lie  free  in  the  space  formerly  occupied  by  the  cell,  or  they 
find  their  way  into  lymph  spaces.  The  nucleus  is  more  resistant 
than  the  cell  body  and  keeps  its  form  and  staining  properties  while 
the  cell  is  still  filled  to  bursting  with  hyahn  bodies. 

The  dissolution  stage  of  the  plasma  cell  nucleus  has  not  been 
observed. 

Mast-cells. — In  the  study  of  mast-cells,  Unna's  polychrome 
methylene  blue  and  carbol-toluidin  blue  have  been  used  to  stain 
the  specific  granules.  The  question  arises  as  to  whether  all  cells 
whose  protoplasmic  granules  have  a  specific  affinity  for  the  red  in 
these  solutions  should  be  considered  mast-cells.  I  have  not  arrived 
at  a  satisfactory  conclusion  on  this  point,  but  for  the  sake  of  con- 
venience it  seems  justifiable  to  consider  them  in  one  group.  As  seen 
in  the  tissues  under  discussion,  they  fall  into  the  following  classes: 


142  Contributions  to  Medical  Science 

1.  The  leukocytic  mast-cell. — These  occur  especially  in  the 
immediate  vicinity  of  blood-vessels,  about  which  they  may  be 
closely  packed.  They  measure  from  five  or  six  to  ten  or  twelve 
microns  in  diameter;  are  spherical  or  cuboidal  in  shape;  have  a 
relatively  large,  intensely  staining  nucleus,  from  three  to  six 
microns  in  diameter,  and  a  number  of  small  protoplasmic  granules, 
which  are  stained  bright  red.  A  single  cell  may  have  only  from 
six  to  twelve  granules,  or  it  may  be  densely  packed  with  them. 
In  one  place  two  of  these  cells  were  seen  within  a  blood-vessel. 
Just  outside  were  several  which  had  two  small  nuclei  each;  these 
apparently  were  dividing  cells.  They  were  so  numerous  in  certain 
fields  that  they  gave  a  reddish  tint  to  the  field  with  a  low-power 
magnification.  From  their  size,  their  relation  to  blood-vessels, 
and  their  actual  presence  within  blood-vessels,  the  inference 
follows  that  they  are  of  leukocytic  origin.  We  have  not  been  able 
to  determine  their  fate. 

2.  The  mast-cell  of  connective-tissue  type. — This  is  the  classical 
mast-cell,  spherical,  oblong,  cuboidal,  spindle,  or  grotesquely 
shaped.  Apparently  its  shape  is  determined  by  its  surroundings. 
Probably  the  most  characteristic  feature  is  that  several  processes 
extend  in  three  or  more  directions.  The  spindle  form  is  seen  very 
often.  A  number  of  these  cells  have  been  found  whose  protoplasm 
at  each  pole  dwindled  away  into  long  clear  fibers,  the  number  of 
specific  granules  being  very  few.  The  nucleus  of  this  variety  is 
oval  or  cylindrical,  and  stains  a  pale  blue.  Two  or  three  granules 
of  chromatin  stain  deeply,  and  have  no  characteristic  arrangement. 
It  is  very  similar  to  the  nucleus  of  the  young  connective-tissue 
cell.  This  type  is  found  in  the  papillary  tissue,  and  they  may 
enter  the  prickle-cell  layer;  they  are  also  found  in  definite  masses 
around  hair  follicles  and  sebaceous  glands,  sweat  glands  and  their 
ducts,  and  in  the  connective-tissue  framework  of  muscles  and 
adipose  tissue,  and  imbedded  in  the  walls  of  larger  blood-vessels. 
Their  origin  and  fate  are  alike  obscure.  The  idea  that  they  are 
mucinoblasts  (H.  F.  Harris)  I  am  not  now  able  to  affirm  or  refute. 
On  a  morphological  basis,  some  appear  to  be  transformed  into 
connective-tissue  fibers.  That  they  also  give  off  some  substance 
which  may  be  of  use  to  surrounding  cells  is  shown  by  the  next  type. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  143 

3.  The  mast-cell  which  possesses  a  halo. — Certain  mast-cells 
are  surrounded  by  an  ill-defined,  structureless  zone,  which  is 
tinted  a  homogeneous  faint  red  by  the  polychrome  methylene  blue. 
It  seems  probable  that  the  same  substance  which  in  the  cells 
attracts  the  methyl  red  has  become  diffused  into  the  surrounding 
tissues,  and  retains  the  original  tinctorial  affinity.  This  accounts 
for  the  diffuse  red  color  seen  in  some  fields  where  plasma  cells  are 
numerous. 

4.  The  plasma-mast  cell. — This  name  is  applied  to  certain  cells 
which  possess  characteristics  of  both  the  plasma-  and  mast-cells. 
They  are  spherical,  ovoid,  or  cuboid;  the  protoplasm  densely 
granular,  and  taking  up  both  the  red  and  the  blue  of  the  poly- 
chrome. The  blue  commonly  predominates,  giving  a  violet,  in 
which  a  distinct  trace  of  red  is  visible.  However,  the  proportions 
of  red  and  blue  taken  up  are  inconstant.  They  occur  where  other 
mast-cells  are  found,  mixed  with  the  dense  cell  infiltrate,  and  in 
the  stroma  of  muscle  and  adipose  tissue.  The  nucleus  in  its 
morphology  may  or  may  not  resemble  that  of  the  plasma  cell. 
The  clear  homogeneous  nucleus  seen  in  certain  mast-cells  occurs 
here  at  times.  Often  it  is  entirely  obscured  by  the  deeply  staining 
protoplasm. 

There  seems  to  be  no  clear  relationship  between  plasma  cells 
and  mast-cells.  It  is  certain  that  it  is  not  the  common  fate  of 
either  to  be  transformed  into  the  other. 

The  cellular  nodules. — The  tuberculoid  nodules  and  multinu- 
clear  giant  cells  of  cutaneous  oidiomycosis  possess  few  features 
to  distinguish  them  from  those  of  tuberculosis.  The  nuclei  of 
giant  cells  have  the  same  disposition  as  in  the  latter  disease.  In- 
stead of  tubercle  bacilli,  the  fungus  cells  are  enclosed;  and  it  is 
more  common  to  find  vacuoles  and  leukocytes  in  the  protoplasm. 
It  seems  probable  that  the  vacuoles  are  spaces  formerly  occupied 
by  organisms  or  leukocytes.  The  nodules  may  resemble  closely 
the  tubercles  of  tuberculosis.  Central  necrosis,  however,  is  very 
unusual,  there  is  not  ordinarily  a  well-defined  zone  of  endothelial 
cells,  and  the  whole  mass  is  infiltrated  with  leukocytes;  they  are 
more  vascular  than  those  of  tuberculosis. 

Tubercle  bacilli  have  not  been  satisfactorily  demonstrated  in 


144  Contributions  to  Medical  Science 

any  reported  case,  and  nothing  similar  to  them  has  been  found 
in  the  cases  I  have  studied. 

A  COMPARATIVE  STUDY  OF  THE  ORGANISMS. 

For  consideration  we  have  seventeen  organisms  cultivated 
from  cases  of  oidiomycosis  or  blastomycosis  of  the  skin,  including 
the  cases  in  literature,  the  one  from  Busse's  case  of  Saccharomy- 
cosis  hominis,  the  one  cultivated  by  Curtis  from  myxoma-like 
tumors,  and  the  one  by  Ophiils  and  Moffitt  from  the  so-called 
protozoic  disease.  A  mould-fungus  noted  by  W.  D.  Montgomery 
on  tubes  inoculated  from  lesions  of  the  protozoic  disease  was 
thrown  away  without  being  studied.  Aside  from  this,  there  are 
several  cases  of  skin  oidiomycosis  and  five  of  the  protozoic  disease 
from  which  cultures  were  not  obtained,  but  in  which  the  character- 
istics of  the  organisms  in  the  human  tissue  are  described.  Since 
the  various  organisms  have  not  been  studied  on  uniform  lines 
by  their  discoverers,  the  desired  thoroughness  in  comparison 
cannot  be  realized.  For  example,  the  facts  as  to  indol  formation, 
acid  production,  endogenous  spore-formation  according  to  pre- 
scribed conditions,  details  as  to  fermentation  and  temperature 
limits,  have  not  been  accurately  described  in  reference  to  this 
or  that  organism,  and  often  points  in  morphology  and  modes  of 
reproduction  are  not  always  clear.  However,  differentiation 
and  comparison  may  be  made  with  reference  to  certain  points 
which  are  given  with  fair  uniformity. 

General  cultural  characteristics  and  reactions,  the  morphology 
of  the  organisms  in  tissues  and  cultures,  their  methods  of  repro- 
duction, and  their  pathogenicity  constitute  points  which  may  be 
utilized  in  making  comparisons.  I  shall  refer  to  the  organisms  by 
the  numbers  used  in  the  chart,  where  cultural  characteristics  are 
indicated  briefly;  supplementary  statements,  however,  are  neces- 
sary for  the  sake  of  clearness. 

Most  of  the  organisms  develop  on  artificial  media  in  from  one 
to  two  days  after  inoculation.  Certain  others  require  from  two 
to  seven  days.  It  is  of  interest  that  the  latter  organisms  are 
those  which  are  particularly  prone  to  form  myceHum.  Those 
organisms  which  form  a  smooth,  paste-like  surface  develop  very 


Oidiomycosis  of  the  Skin  and  Its  Fungi  145 

readily;  those  with  irregular  surfaces  and  aerial  hyphae  develop 
more  slowly. 

As  noted,  most  organisms  grow  readily  on  ordinary  media, 
either  at  the  room  or  brood-oven  temperature.  Faintly  acid, 
saccharin,  or  glycerin  media,  or  a  wort  preparation  and  potato 
constitute  a  favorable  media.  In  our  experience  the  maltose  agar 
of  Sabouraud,  made  sHghtly  acid,  is  the  safest  and  surest  single 
preparation.  The  observation  that  mycelium  forms  abundantly 
in  dog's  blood-serum  suggests  that  a  preparation  of  this  material 
may  prove  a  suitable  medium. 

As  to  the  surface  appearance  on  solid  media  the  organisms  fall 
into  three  classes,  of  which  organism  5  is  the  type  of  one,  11  of 
another,  and  12  of  the  third.  The  first  group  presents  in  young 
cultures  elevated,  moist,  soft,  white  colonies  which  soon  coalesce  to 
form  a  fleshy  growth  of  paste-like  consistence.  The  second  type 
at  first  presents  a  granular  surface,  sHghtly  elevated,  and  as  the 
colonies  coalesce  the  elevation  becomes  more  pronounced,  the 
growth  incorporates  itself  with  the  medium,  and  eventually  the 
surface  appearance  is  that  of  a  piece  of  crumpled  cloth  or  a  niass  of 
coiled  earth-worms.  The  third  type  is  represented  by  those  fungi 
which  produce  aerial  hyphae,  the  surface  being  white,  dry,  and 
flour-like,  or  exhibiting  hyphae,  which  eventually  cover  the  inner 
surface  of  the  tube.  Those  following  the  first  type  are  easily  broken 
up  and  smeared  over  the  surface.  Those  included  in  the  second 
and  third  types  are  firmly  incorporated  with  the  medium  and  can 
be  broken  up  only  by  loosening  fragments  of  impregnated  sub- 
stratum. The  surface  of  these  organisms  in  the  first  type  becomes 
dirty  or  slightly  brown  with  age.  This  also  shows  through  the 
reverse  surface  of  the  tube.  The  reverse  surfaces  of  the  second 
and  third  also  show  color  changes,  the  second  becoming  a  dirty 
brown,  and  the  third  exhibiting  a  rich  golden-brown  color,  like 
that  of  a  well-colored  meerschaum.  The  organisms  of  the  first 
type  primarily  send  segmented  hyphae  into  the  substratum; 
this  property  is  lost  eventually  during  the  artificial  life  of  the 
organism,  only  a  surface  growth  appearing.  The  second  and  third 
groups  persistently  infiltrate  the  substratum.  In  liquid  media, 
also,  the  same  groups  produce  three  corresponding  appearances. 


146  Contributions  to  Medical  Science 

The  first  occurs  in  the  bottom  as  a  flocculent  sediment,  the  super- 
natant fluid  remaining  clear;  shaking  disseminates  the  growth 
and  gives  the  medium  a  milky  appearance.  The  second  type, 
which  is  strongly  mycelial,  produces  a  membranous  top  growth, 
and  in  addition  coherent  masses  or  tufts  of  mixed  hyphal  and 
spherical  elements  which  lie  in  the  bottom  of  the  tube  and  adhere 
to  the  sides.  As  the  top  membrane  becomes  more  dense  it  sinks 
to  the  bottom  and  a  new  membrane  develops.  The  organisms 
with  a  pronounced  aerial  growth  form  at  first  a  single  coherent 
mycehal  tuft.  After  two  or  three  weeks,  because  of  the  separation 
of  spores  and  segments,  other  small  tufts  appear.  A  top  growth 
is  not  formed,  and  the  intermediate  fluid  is  unclouded.  Likewise 
on  gelatin  or  agar  plates  the  three  groups  remain  distinct.  The 
first  forms  closely  granular  colonies,  the  individual  cells  of  which 
can  be  seen  with  a  low-power  objective.  Films  from  such  colonies 
show  only  a  mixture  of  spherical  and  elongated  cells.  To  the 
naked  eye  they  appear  as  round,  white,  moist,  elevated  points, 
and  the  deeper  colonies  are  much  smaller  than  those  on  the  surface. 
The  second  type  produces  colonies  which  to  the  naked  eye  are 
somewhat  glistening.  Under  a  low  power  they  are  seen  to  consist 
of  radiating,  segmented,  and  branched  hyphae,  the  contour  of  the 
colony  being  almost  circular.  The  third  type  produces  colonies 
of  a  feathery  naked-eye  appearance,  the  processes  being  of  un- 
usual length.  Under  the  low  power  this  is  seen  to  be  made  up  of 
branching  hyphae  which  are  closely  segmented  and  beset  with 
numerous  lateral,  unicellular  offshoots.  As  the  hyphae  reach  the 
surface  a  moulded  appearance  develops. 

Organisms  11  and  13  (see  comparative  table)  produce  acetic 
acid. 

Organisms  3,  4,  6,  and  12  possess  no  fermenting  powers.  A 
large  percentage  ferment  glucose  and  maltose  solutions.  Organism 
2  is  unique  in  being  able  to  invert  and  ferment  saccharose  and  to 
ferment  lactose  solutions.  In  all  cases  where  fermentation  results 
CO2  and  alcohol  are  formed;  two  organisms  only  produce  acetic 
acid  in  the  process. 

Organism  10  is  unique  in  its  ability  to  produce  indol,  using 
Theobald  Smith's  method. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  147 

All  are  obligate-aerobic  in  their  growth,  but  No.  2  existed  in 
a  latent  state  for  a  number  of  days  without  the  presence  of 
oxygen. 

Freezing  has  not  killed  those  organisms  with  which  the  experi- 
ment has  been  tried;  the  thermal  death  point  is  in  the  neighbor- 
hood of  45°  to  58°  C,  for  from  two  to  five  minutes.  They  resist 
desiccation. 

Explanations. — Cases  are  referred  to  by  the  names  of  the 
observers;  and  those  composing  the  present  series,  by  the  Roman 
numerals  used  in  preceding  pages. 

The  numbers  indicate  chronological  order  (except  17),  and 
correspond  to  numbers  used  in  ''Comparative  Analysis  of  the 
Organisms,"  p.  144. 

Letters  refer  to  notes  on  following  page. 

The  signs  for  the  most  part  are  self-explanatory. 

+ :  affirmative. 

—  :  negative. 

H — ,  or  — f- :  inconstant,  or  in  moderate  degree. 

? :  not  clear,  or  satisfactory. 

Blank  squares:   no  information  at  hand. 

la.  Buschke  observed  that  short  threads  sometimes  formed. 

ih.  Demonstrated  by  Busse,  using  H.  Moeller's  method. 

2a.  Does  not  grow  on  blood-serum. 

2h.  Not  freely. 

2c.  Noted  by  Anna  Stecksen. 

3a.  When  myceUum  ceased  to  form,  the  surface  appeared 
gelatinous. 

36.  This  is  not  mentioned  by  Gilchrist,  but  Buschke  observed 
it  in  cultures  sent  to  him  by  Gilchrist,  who  observed  only  a  prickly 
surface. 

3c.  The  maximum  Hmit  for  growth  is  40°  C. 

4a.  In  a  few  days,  as  stated  by  Hessler.  In  this  laboratory 
its  development  is  noted  after  twenty-four  hours. 

46.  A  "little  smaller  than  Gilchrist's"  (Hessler). 

5a.  Observed  recently. 

6a.  Through  the  medium  of  a  guinea-pig  which  had  been 
inoculated  with  fresh  tissue. 


Is 

Si 

a 

3 
□ 

3  3 

O  " 

tumefaciens 
Oidium 

dermatitidis 
Oidium    Hek- 

'c 

a  = 

.2-" 

5^ 

S2 

Oldium      Hek- 

toenii  II(io,o) 
Oidium 

aceticum 
Oidium 

hyphomycoide 
Oidium 

hyphomycoide 
Oidium 

hyphomycoide 
Oidium 

hyphomycoide 
Oidium 

hyphomycoide 

Terms  Which 

Have  Been 

Applied 

TO   THE 

Various 
Organisms 

S3 
11 

«  2  K 

E2§ 
m 

.2 

toT3 

n 

5 

STVHINV   01  AiDINaOOHXVJ 

+T 

+ 
+ 

+ 

A, 

+  ;  + 

+ 

o 

§ 

ei 

B< 

H 

Pi 

"3 

u 

3 
1 

saiodsoDsy 

1  1 

1 

1 

1 

-'  '  1 

+     +     +     +     ^ 
1        1        1        1 

Btprao3  jEiajE-j  1    1   1 

1 

1 

1 

-x.  1    1   + 

+     +     +     +     ^ 

cd  to 

II 

3   3 

sajodsopng      | 
JO  sajodsoDsy  | 

1 

1 

^1         (V.       (V. 

+  '   +  + 

ru       ru       ru       fu 

+   +   +   + 

■Eiprao3  iBurtn 

-J3X  JO    IBJ3JB7 

+    + 

+ 

- 

-+I  + 

+  +  +  + 

nouBjuara 
-Sa's  I^qdXH 

1 
+ 

+    + 

+ 

- 

++  +  + 

+  +  +  + 

aaippng  |  ++ 

+    + 

+ 

+ 

+  +++  +  + 

+  +  +  + 

3 

saiodsopnj 

1"-- 

(-c        f\- 

- 

ru 

'^"'  ""  + 

^         ^         A-         ft.            , 

+        +        +        +        + 

noijB^uaniSas 
q^iM  SBqdXjj 

II       III                l'^    1     1     1     1     1     1 

Stnppng  |  ++ 

+    + 

+ 

+ 

++^  +  + 

+        +        +        + 

>> 
o 

s 

o 

n 

o 

1 

§■ 
2 

(I  )saiodsopn3-opn3sj 

+ 

+ 

*l   +  + 
++  +  + 

+        +        +        + 
+        +        +        + 

snapn^ 

+ 

1 

1 

/%-      '^      '^ 

1     1 

ri.       ri.       A.       n. 

1    +    1     1 

saionD^A  |  ++ 

+    + 

+ 

+ 

+  +  + 

+  +  +  + 

M 

iBfnu'BiQ  Xiasj'BOQ  |  +-|- 

+   + 

+ 

+ 

+  +  + 

+  +  +  + 

h4 

jBinireio   /{pnij  |  ++ 

+    + 

1 

+ 

+  +  + 

+  +  +  + 

^ 

"3 

a 
c3 

51 

S3jniin3  1  ++         1              1                            1       1       1       1       1       1       1                1 

snssix 

++ 

+ 

1 

+    1 
^    1    + 

li  — 

■& 

2 

Oc 

S3inipi3 

1  1 

++ 

+    + 

+ 

+ 

+++  +  + 

+  +  +  + 

anssix  1  ++ 

+    + 

+ 

+ 

++<^.       +       + 

+  +  +  +  + 

acqdXH 
pajmof 
paqDUBjg 

s3Jnipio  ni 

5J 

+    + 

+ 

++       +       + 

+  +  +  +  + 

anssjx  HI  1    1    1 

1      1 

1 

r^        1         1 

1     1     1     1     1 

o  o 

?r 

to  ro 

o 

I 

HI          .O 

1 

O        >o       O 
t<5        O         O 

o      o     -o      o      o 

MM                           M 

PAO 

S3IIHP13  ni  1  ++ 

+         + 

+ 

+ 

++      +      + 

+    +    +    4- 

snsstx  ni  |    | 

1 

1 

A.          1           1 

1      1      1      1 

fBDuaqds 

saiTupio  ni  1  ++ 

+ 

+ 

+ 

+++       +       + 

+    +    +    + 

snssjx  11  1  ++ 

+         + 

+ 

+ 

++--       +       + 

+    +    +    +    + 

ONiAHa  xa  aaruji  |                 1       1       1                           III                                          | 

SXD 

aHX 

;moj-qjB3a 

« 

Snizaai J  Xq  paiirj  | 

1 

1     1 

oiaoHSV  aivonao  |  ++ 

+         + 

+ 

+ 

+  +  + 

a: 

o 

1 

1 

2 

Oh 

PPV  apaay  |      + 

1 

1 

1    +    1 

joqoDiv  1  ++ 

+ 

+ 

+  +    1 

'OD  1  ++ 

+ 

+ 

+  +    1 

9sopbt|    :^      I     I     I     I             III 

asojiBj^  1      + 

+ 

+ 

+  +    1 

asoanio  |  ++ 

1            + 

^ 

1 

+  +    1 

asoi'BqDD'BS  JO 
nopBinanuaj  pnB  uoisjaAnj 

+                111                                                  III 

|.« 

to           ■* 

u> 

vO 

f^        00    O*  O           M           C» 

W            M            M 

to        •*        V)      \o         t^ 

nopBinaBoo  I    I       I   I    I   I 


P-iinsN  I  +        +++ 


ann^^nv 


I    I 


PPV  I   I       III 


l  +  l 


+  1    I 


I   1  + 


1  + 


NOixo0aoH<j-ioaNi  |  |   |   | 


+  1   I 


urjBiao 


uoiiDEjanbii      |   |   |    |   |   | 


sai^iJ 


spwiqx  anrjBtpB^  {    |      +  |    | 


jBinuwo  I  +        ++ 


5  » 

3  2 
pqO 


pmij  in^jBOjadns  i^3\0  \     +++++ 


sjpx  ?uaj3qo3  |       I  +  II    I 


inamtpas  luajnDDOij  |  ++  |  +++ 


q;/ttoio  dox  |  +  I   I    I   I    I 


uoij'BogijDtuj  puay 


IT  I    I    I 


Xq  taiU'Bjjsqns  )0  noi)'Ej:)aaaj 


+  1 
1  + 


aSy  q?iM  SuiuMOjg     +- 1-     -|-4- 


J01O3  iCjJTQ  [     I     I 


mmpaj^  qjiM  pajwod 
-jODaj  puB  5uaiaqo3 


iCiSBjj 


I    1  + 


++T+++ 


ajnjsiOK    ++++++ 


snoissdjdaQ  a^fii-jaj'Ej^  I 


++ 


suoiiEAaia  intoj-aT<j  |  +     ++ 


qjoouis  JsiTj  ;v  I  ++  I  +++ 


l  +  l 


I++ 


++I 


+++++++ 


+++++++ 


+  1  I  I  I  I  I 


1  +  I  I  I  I  I 


a+++++* 
I  I ++++++ 


++++++ 


+  1 


3WM I ++++++     +1 


++++++ 
++++++ 


+  1    I    I    I    I    I 


++I    I    I    I    I 


+  1    I    I    I    I    I 


+  1    I 


I    I 


+ I +++++ 


pai^Aaja  I  ++++++  ++' 


«  B.  tl  "^ 


anuBqoDBS 


XjBnTpjo|j-+++++_ 


IBijnaN 


anjiBJuv 


PPV    ++       + 


saanxTOO  io  aoraaj  NoixvanoNi    c^      i   i 

Q   " 


+++++++ 


++ 


I  I 

+ 


+  1 
++ 


I  I  I  LJ  I 


siomnx  snoi-BuioxXj^  |    I  +  I    I    II       I    I    I    I       I    I    I    I 


snj     +    +++  I      +A.  I  + 


anssjx  1  +  I  +    4-" 


-++ I  =+ I   1  + 


iH  «  »0  ^  >riO  «J  a  <S  O  I 


TO  ^  >0*0  r^ 


*  'r  h  **  fft  O  S^ 


„s>>^>^>« 


t/i  k-  o  cA^  S^  rt-—  ^  v\  <fi  m  tfi  xf)  tn  ViJ^ 

eQUOKBOPQ(J5Quuuouuuo 


I50 


Contributions  to  'Medical  Science 


ya.  The  characteristics  of  this  organism  have  not  been  pub- 
lished.    The  observer's  description  is  lacking  in  details. 

8a.  Description  of  biological  features  altogether  lacking. 

ga.  Detailed  description  not  yet  pubHshed. 

9&.  Gilchrist  noted  that  the  organism  formed  a  branched, 
jointed  myceUum,  "which  frequently  contained  spores." 

loa.  O.  Hektoenii  II  differs  from  O.  Hektoenii  I,  in  producing 
indol. 

iia.  Short  prickles  cover  surface  of  growth. 

iih.  Slowly. 

12a.  At  first  incorporated  with  medium;  later  an  aerial  growth 
occurs. 

12&.  Colorless  until  white  aerial  hyphae  appear. 

I2C.  Reverse  surface  of  slants  assumes  rich  golden-brown 
color  in  time. 

13a.  From  myxomatous  nodule.  Organism  obtained  recently; 
full  cultural  and  pathogenic  properties  not  worked  out  yet. 

14a,  15a,  i6a.  Recently  obtained.  Full  cultural  and  patho- 
genic properties  not  worked  out. 

166.  Simply  stated  that  the  organism  is  a  mould-fungus. 

The  organisms  in  the  tissues. — In  the  tissues  of  all  cases  of 
blastomycetic  dermatitis,  the  appearances  of  the  various  organisms 
show  much  similarity.  The  common  features  are :  a  homogeneous, 
doubly  contoured  capsule,  a  protoplasm  which  is  finely  and  coarsely 
granular,  often  vacuolated,  and  the  occurrence  of  pairs  or  budding 
forms.  Between  the  protoplasm  and  capsule  a  clear  zone  of 
varying  thickness  is  often  found.  The  protoplasm  may  be  nearer 
the  capsule  at  one  pole  than  at  the  opposite,  in  which  case  the 
clear  space  varies  correspondingly  in  thickness.  In  buds  also 
it  is  more  delicate  than  in  the  mother  cell.  While  it  appears  due 
to  shrinking  of  the  protoplasm  in  some  instances,  the  fact  that  a 
similar  condition  is  sometimes  seen  in  water  mounts  of  fresh 
tissue  makes  it  probable  that  the  clear  space  is  a  structural  entity. 
However,  it  is  not  present  constantly  in  fixed  sections,  mounts 
from  fresh  tissue,  or  cultures.  The  capsule  proper — memhrana 
propria — always  present  in  the  tissue,  presents  an  inner  and  an 
outer  contour,  and  appears  homogeneous.     Its  chemical  nature 


Oidiomycosis  of  the  Skin  and  Its  Fungi  151 

is  not  known;  experimenters  have  not  been  able  to  demonstrate 
a  cellulose  constituent.  In  adult  spherical  cells  its  thickness  is 
uniform,  but  in  budding  and  constricted  forms  it  becomes  nar- 
rower on  the  bud  and  in  the  constriction.  In  fixed  and  dehydrated 
tissue  it  may  present  numerous  fractures.  It  has  no  charac- 
teristic staining  affinities;  in  hematoxylin  and  eosin  sections 
it  may  stain  deeply  with  eosin,  but  more  commonly  both  stains 
are  taken;  in  methylene  blue  preparations,  differentiated  in 
glycerin-ether  mixture,  it  is  colored  a  deep  blue;  in  certain  sections 
it  has  taken  up  acid  orcein  deeply,  and  with  proper  manipulation 
it  may  be  made  to  stain  with  carbol-f uchsin ;  it  is  usually  decolor- 
ized by  Gram's  method,  but  may  stain  deeply  with  Weigert's 
modification.  The  protoplasm  is  central  and  spherical,  except  in 
budding,  constricted,  or  indented  forms,  where  it  conforms  its 
shape  to  that  of  the  capsule.  The  structural  elements  so  far 
recognized  are  the  spongioplasm,  small  and  large  granules,  and 
vacuoles.  The  spongioplasm  is  seen  in  thin  sections  stained  with 
eosin,  methylene  blue,  or  orcein,  but  only  in  certain  cells;  many 
stain  so  deeply  and  diffusely  that  no  structure  can  be  made  out. 
It  consists  of  various  sized  anastomozing  fibers,  which  are  continued 
on  the  surface  of  the  protoplasm,  where  they  unite  to  form  a 
delicate  homogeneous  envelopment  of  that  substance.  Usually 
clear  unstained  spaces  lie  in  the  fiber  meshwork;  it  is  possible  that 
when  the  protoplasm  stains  diffusely,  chromophilic  granules 
occupy  these  spaces. 

Small  granules  are  seen  invariably,  except  in  degeneration 
forms,  where  nothing  but  the  capsule  remains.  They  stain  well 
with  nuclear  dyes  and  have  no  characteristic  distribution.  In 
addition,  there  are  the  larger  granules,  or  rather  spherules,  which 
have  been  mentioned  in  connection  with  some  of  the  cases.  They 
occurred  also  in  the  case  studied  by  Hyde  and  Hektoen,  in  that  of 
Anthony  and  Herzog,  in  that  reported  by  Coates,  and  in  F.  G, 
Harris'  case.  There  may  be  two  or  three  to  eight  or  ten  in  a  cell; 
they  are  perfectly  spherical,  and  are  often  distributed  evenly  in 
the  periphery  of  the  cell.  They  are  rarely  more  than  0.5  micron 
in  diameter,  and  stain  with  nuclear  dyes.  In  the  last  case  of  this 
series  these  spore-like  bodies  are  sometimes  i  to  1.5  microns  in 


152  Contributions  to  Medical  Science 

diameter,  from  four  to  eight  occurring  in  a  cell.  They  stain  best 
with  hematoxylin,  and  are  found  in  a  relatively  small  number  of 
cells.  In  Case  IV  they  are  seen  in  one  instance  lying  partly  out- 
side a  cell  with  a  ruptured  capsule.  The  conception  that  they 
are  endospores,  however,  finds  no  satisfactory  proof.  No  transi- 
tion forms  have  been  found.  Perhaps  they  are  either  spores  or 
represent  a  fragmented  nuclear  substance.  In  the  abscesses  and 
granulation  tissue  are  seen  similar  bodies,  which  are  extracellular 
and  appear  to  be  a  product  of  tissue  cell  degeneration. 

Vacuoles  have  been  seen  in  the  organisms  of  all  cases.  Com- 
monly, only  one  is  seen  in  a  cell,  but  there  may  be  two  or  three; 
or  the  whole  protoplasmic  body  may  be  occupied  by  several, 
which  are  outlined  by  fine  granules  over  their  surface.  They  are 
perfectly  clear,  sharply  defined,  and  one  may  connect  with  another 
by  a  small  neck.  Rarely  they  occur  in  buds.  A  nucleus,  in  the 
ordinary  sense  of  the  term,  has  been  demonstrated  in  no  case. 

Degeneration  or  involution  stages  are  represented  by  empty 
capsules,  and  by  cells  with  an  indented  capsule  and  protoplasm, 
having  a  sickle  shape. .  Empty  capsules  may  stain  as  densely  as 
those  enclosing  a  chromophilic  protoplasm. 

An  additional  capsular  structure  has  been  seen  in  a  few  cases. 
It  has  been  termed  adventitious  or  accidental.  It  lies  outside  the 
membrana  propria,  is  irregularly  laminated,  and  its  surface  often 
frayed  moderately;  it  stains  bluish-green  with  the  polychrome 
methylene  blue,  and  sometimes  deeply  with  eosin.  It  may  be  due  to 
a  secretion  of  the  parasite,  or  may  be  due  to  the  chemical  effect 
of  the  organisms  on  tissue  fluids.  This  structure  was  conspicuous 
in  Gilchrist's  second  case,  and  in  one  of  this  series  (IV) .  In  a 
group  of  organisms  it  apparently  plays  the  part  of  a  cement  sub- 
stance. Busse's  organism  from  the  case  of  Saccharomycosis 
hominis  and  that  of  Curtis  from  myxoma-like  tumors  possessed  the 
adventitious  capsules;  in  other  respects  their  structure  is  similar 
to  the  type  described.  These  observers  also  found  the  adventi- 
tious capsule  in  old  cultures. 

The  organisms  seen  in  the  so-called  protozoic  disease  are  larger 
than  those  of  blastomycetic  dermatitis  and  their  protoplasm 
becomes  broken  up  into  fifty  or  a  hundred  spores,  which  are  liber- 


Oidiomycosis  of  the  Skin  and  Its  Fungi  153 

ated  through  a  break  in  the  maternal  capsule.  From  transition 
forms  observed,  the  writers  conclude  that  the  spores,  either  within 
the  mother  cell  or  after  escaping  from  it,  grow  and  slowly  acquire 
adult  characteristics.  The  capsule  is  doubly  contoured.  Budding 
cells  and  an  adventitious  capsule  have  not  been  described.  Spheri- 
cal cells,  taken  directly  from  human  tissue  and  mounted  in  a 
nutrient  hanging  drop,  immediately  sprout  mycelial  threads 
(Ophiils  and  Mofl&tt) .  This  experiment  was  repeated  from  inocu- 
lated animals.  In  only  one  of  the  organisms  I  have  studied  were 
similar  appearances  observed. 

Proliferation. — All  organisms  of  blastomycetic  dermatitis,  and 
those  of  Busse  and  Curtis,  mentioned  above,  while  in  the  parasitic 
state,  proKferate  by  the  gemmation  of  capsulated  spherical  cells. 
It  is  probable  that  they  are  a  fructification  form;  i.e.,  spores,  in 
the  sense  in  which  the  term  is  applied  to  such  cells  in  oidia  and 
allied  organisms.  In  the  meantime,  the  position  of  the  endo- 
cellular  bodies  of  the  organisms  of  protozoic  dermatitis  as  spores 
must  be  held  suh  judice;  the  same  applies  to  similar  bodies  seen  in 
organisms  from  the  cases  of  this  series. 

Hyphae  have  not  been  described  in  the  tissues  of  oidial  derma- 
titis, the  protozoic  disease,  or  Saccharomycosis  hominis.  A 
pseudo-mycelial  formation  was  seen  in  an  abscess  communicating 
with  the  surface  in  one  of  the  cases  here  described;  there  were 
several  chains  of  six  to  eight  spherical  cells  in  each;  they  did  not 
branch. 

The  steps  in  the  budding  process  have  been  described  by 
Gilchrist,  Hektoen,  and  others;  they  seem  to  be  the  same  in  tissue 
and  in  cultures.  The  membrana  propria  becomes  thin  at  one  point 
and  is  pushed  out  in  the  form  of  a  bud  by  a  process  of  protoplasm 
and  its  clear  space.  The  mass  of  protoplasm  in  the  bud  is  at 
fiirst  structureless;  it  gradually  increases,  the  capsule  and  clear 
space  become  wider,  and  the  protoplasm  eventually  acquires 
adult  characteristics.  Rarely  it  becomes  vacuolated  before  detach- 
ing itself.  Separation  is  accomplished  by  union  of  the  common 
capsular  substance  at  the  point  of  constriction  between  the  two 
cells.    A  cell  is  sometimes  found  with  two  or  three  buds  attached. 

In  the  lesions  the  parasites  measure  from  10  to  17  microns  in 


154  Contributions  to  Medical  Science 

diameter;  exceptional  cells  may  be  larger,  and  others  are  seen 
measuring  less  than  lo  microns.  In  the  dermatitis  of  Wernicke's 
disease  the  organisms  are  i6  microns  or  more  in  diameter. 

Morphology  of  organisms  in  cultures. — In  cultures  all  organisms 
have  morphological  variations  not  seen  in  tissues,  and  each  may 
present  two  or  more  varieties  of  cells.  The  spherical  (sometimes 
oval)  budding  cell  is  a  common  feature  of  all  the  organisms  in 
cultures.  Also  the  formation  of  jointed  hyphae  has  been  noted 
in  all  organisms  thoroughly  described.  In  some  cases  in  the 
chart  this  process  is  not  mentioned,  but  it  is  to  be  noted  also  that 
many  other  essentials  are  not  given.  The  proportion  of  spherical 
(or  oval)  budding  cells  to  mycelium  varies  greatly  in  the  different 
organisms,  and  in  the  same  organism  at  different  periods.  Either 
one  or  the  other  may  disappear  almost  entirely  during  artificial 
life.  Their  relation,  however,  is  fairly  constant  in  the  same 
organism,  and  in  comparing  with  other  organisms  this  feature  is 
of  differential  value.  Those  described  by  Hektoen,  Gilchrist,  and 
Stokes,  and  the  organism  from  Case  I  at  first  exhibited  abundant 
mycelium.  In  this  organism  mycelial  formation  soon  ceased, 
but  it  was  reinduced  by  inoculations  into  mice.  On  the  other 
hand,  the  organism  from  Case  III  at  first  formed  spherical  (or 
oval)  cells  and  mycelium  about  evenly;  now,  however,  the  growth 
is  almost  entirely  mycelial.  Those  organisms  which  produce 
mycelium  ordinarily  give  off  lateral  or  sometimes  terminal  conidia 
which  are  usually  sessile,  but  may  be  pedunculated. 

As  described  below,  aerial  hyphae  formed  constantly  in  the 
organisms  for  five  cases,  and  exceptionally  in  a  sixth.  They  are 
finer  than  the  submerged  hyphae,  are  irregularly  segmented, 
produce  lateral  pedunculated  spherical  conidia  in  abundance,  and 
rarely  terminal  ascospores  ( ?) .  On  solid  media,  mycelium-forming 
organisms  send  their  hyphae  into  the  substratum.  In  all  cases 
the  hyphae,  aerial  or  submerged,  are  segmented  and  branch  freely. 

All  organisms,  whatever  the  form,  possess  capsules,  which  may 
be  delicate,  or  thick  enough  to  show  a  double  contour;  in  general, 
they  are  thinner  under  artificial  conditions  than  under  parasitic. 
A  granular  condition  of  the  capsule  is  sometimes  brought  out 
by  hematoxylin  or  methylene  blue;    otherwise  it  appears  homo- 


Oidiomycosis  of  the  Skin  and  Its  Fungi  155 

geneous  and  is  very  refractive.  A  clear,  structureless  space  is 
often  seen  between  the  capsule  and  protoplasm,  but  it  is  incon- 
stant. It  appears  to  represent  a  watery  substance  between  the 
protoplasm  and  the  capsule. 

The  protoplasm  follows  the  contour  of  the  capsule.  In  un- 
stained specimens  it  may  be  clear  or  may  be  differentiated  into 
stationary  or  motile  granules,  peculiar  refractive  spherules,  vacuoles, 
and  a  nucleus-like  structure  which  possesses  a  faint  pink  or  orange 
tint.  Stained  specimens  show  a  dehcate  membrane  surrounding 
the  protoplasm;  this  is  the  structure  mentioned  above  which  is 
continuous  with  the  spongioplasm.  The  latter  structure  shows 
poorly  in  fixed  and  stained  cover-glass  preparations.  It  is  not 
known  whether  or  not  the  stationary  granules  are  of  the  same  nature 
as  the  motile.  The  latter  are  seen  in  mounts  in  water,  and  are 
brought  out  more  clearly  with  neutral  red  as  a  vital  stain;  very 
dilute  fuchsin  or  hematoxylin  solutions  answer  the  same  purpose, 
without  fixing  the  granules  immediately.  The  motion  is  of  an 
exaggerated  Brownian  type.  Not  all  motile  granules,  however, 
are  stained  in  this  way,  and,  on  the  other  hand,  certain  stationary 
ones  take  up  the  stains.  I  have  not  been  able  to  determine  whether 
these  bodies  are  foreign  or  a  part  of  the  cell  organism.  The  latter 
is  probably  the  correct  conception;  and  they  no  doubt  correspond 
to  Raum's  "Granula,"  in  part  at  least.  In  fixed  and  stained 
preparations  the  chromophilic  granules  are  irregularly  distributed. 
Some  cells  show  absolutely  none.  They  are  sometimes  grouped  in 
semilunar  shape  in  the  periphery  of  the  protoplasm,  resembling 
a  nuclear  structure.  Apparently,  however,  other  structures  than 
the  granules  mentioned  are  capable  of  taking  up  stains  in  fixed 
preparations,  as  the  volume  of  stained  protoplasm  may  be  in  great 
excess  of  the  granules  seen  in  fresh  cells.  Young  buds  and  the 
growing  ends  of  hyphae  usually  have  an  undifferentiated  proto- 
plasm. As  the  bud  approaches  the  size  of  the  mother  cell,  and  as 
the  hyphae  become  segmented,  granules  and  the  nucleus-like 
structure  are  acquired. 

We  have  been  able  to  form  no  clear  idea  of  the  nature  and 
function  of  this  nucleus-Hke  structure.  It  may  or  may  not  be 
present  in  active  adult  cells  (isolated  or  myceUal);   but  is  absent 


156  Contributions  to  Medical  Science 

uniformly  in  very  young  cells  and  in  degeneration  or  involution 
forms.  It  has  a  faint  pink  color,  may  be  spherical  or  oval,  presents 
no  visible  structure,  and  has  not  been  differentiated  by  staining 
methods  at  our  command.  In  spherical  or  oval  cells  it  is  always 
spherical,  and  in  the  latter  form  occupies  the  smaller  pole.  It 
varies  in  size  from  0.5  or  i.o  micron  to  one-quarter  or  one-third 
the  diameter  of  the  whole  cell.  It  occurs  in  all  culture  media,  but 
in  greatest  numbers  in  cells  grown  on  a  moist  gypsum  block. 
Occasionally  there  are  two  in  a  cell,  one  at  either  pole.  Rarely 
there  is  a  single  large  body  in  the  center  of  the  cell.  Hyphal 
segments  may  contain  one  or  several  such  bodies,  of  oval  or  spheri- 
cal shape,  and  they  likewise  occur  in  unsegmented  threads.  As 
to  function,  it  is  possible  that  they  are  involved  in  reproduction; 
but  as  proliferation  seems  to  occur  extensively  without  their 
presence,  this  could  be  only  a  facultative  office. 

We  cannot  consider  them  nuclei  as  the  term  is  understood 
usually;  however,  they  may  represent  an  analogous  substance, 
the  constant  presence  of  which  is  not  essential  to  cell  hfe.  Simi- 
larly, their  structure  precludes  their  being  considered  true  spores. 

Clear,  structureless,  highly  refractive  spherules  occur  in  the 
seven  organisms  we  have  studied.  In  three,  those  of  Hektoen, 
Hessler,  and  that  from  Case  I,  which  are  almost  identical  mor- 
phologically, these  bodies  are  inconspicuous,  and  occur  for  the 
most  part  when  the  organisms  exist  under  unfavorable  conditions. 
They  are  not  numerous  and  may  not  be  related  to  similar  bodies 
which  occur  freely  in  the  organisms  from  other  cases  when  the 
latter  are  growing  luxuriantly.  Perhaps  the  bodies  are  the  result 
of  plasmolysis  due  to  osmotic  phenomena. 

Plates  7  and  9,  Figs.  12,  i8a,  and  186,  illustrate  the  spherules 
under  consideration  as  seen  in  the  organisms  referred  to.  They 
appear  of  similar  nature  in  all,  are  from  one  to  three  or  four  microns 
in  diameter,  and  seem  to  be  structureless.  There  may  be  only 
a  few  in  a  cell,  or  the  latter  may  be  completely  filled.  In  no  case 
have  they  been  observed  to  escape  through  the  capsule  or  acquire 
definite  structure.  It  has  been  mentioned  that  they  occur  in 
nearly  all  cells  of  organism  from  Case  VII,  as  it  was  observed  in 
the  lung  of  a  dog.       Similar  bodies  were  found  extracellular,  but 


Oidiomycosis  of  the  Skin  and  Its  Fungi  157 

did  not  develop  into  a  recognizable  fonn  of  the  fungus.  Further- 
more, cells  filled  with  them,  when  mounted  in  a  hanging  drop, 
sprouted  mycelium,  the  spherules  apparently  taking  no  part  in 
the  process  and  gradually  disappearing.  Their  function,  and 
the  conditions  of  their  appearance  and  disappearance,  are  not 
clear.  Their  spore  nature  seems  probable,  but  as  proof  is  lacking, 
the  subject  must  be  referred  for  further  study. 

In  general,  vacuoles  seem  to  occur  as  an  expression  of  unfavor- 
able surroundings,  or  as  an  indication  of  age,  degeneration,  and 
involution.  All  organisms  which  have  existed  for  a  few  days  on  a 
moist  gypsum  block  at  room  temperature  show  a  large  percentage 
of  vacuolated  cells.  The  same  is  found  on  quite  old  culture 
tubes.  On  the  other  hand,  vacuoles  are  not  incompatible  with 
active  vegetative  growth,  as  they  are  often  seen  in  maternal  budding 
cells.  Usually  not  more  than  one  is  seen  in  a  cell  (as  in  tissues), 
but  there  may  be  two  or  three  or  the  whole  protoplasmic  space  may 
be  occupied  with  them. 

As  degeneration  forms  in  cultures  we  recognize  large  cells, 
often  with  thick  and  irregular  capsules,  whose  protoplasm  is 
occupied  by  vacuoles  or  finely  granular  material.  Mycelial 
segments  are  found  in  a  similar  condition.  Empty  and  some- 
times fragmented  capsules  are  seen.  If  a  plate  or  stroke  culture  is 
made  from  a  tube  where  these  forms  constitute  almost  the  whole 
growth,  an  unexpectedly  liberal  culture  results.  This  seems  to  be 
evidence  that  many  cells,  apparently  degenerated,  may  again 
vegetate  actively  under  favorable  conditions,  i.e.,  they  are  involu- 
tion forms. 

The  organisms  have  not  been  found  to  possess  differential 
staining  reactions.  Those  from  cultures  stain  more  deeply  and 
with  a  greater  variety  of  dyes  than  those  in  tissue.  In  general, 
young  and  vegetating  cells  stain  with  all  nuclear  dyes,  resisting 
decolorizing  agents  moderately  well;  while  involution  and  degen- 
eration forms  stain  poorly  or  not  at  all.  Cells  from  cultures  for 
the  most  part  stain  deeply  by  Gram's  method;  in  sections  a  small 
proportion  only  retains  the  stain.  The  refractive  spherules  seen 
in  the  cells  in  cultures  are  not  brought  out  clearly  in  stained  films. 
The  occurrence  of  chromophilic  spherules  under  parasitic  condi- 


158  Contributions  to  Medical  Science 

tions  has  been  mentioned.  Unna's  polychrome  methylene  blue 
has  been  of  the  best  service  in  staining  the  organisms. 

Proliferation  in  cultures. — As  to  proliferation,  the  organisms 
fall  into  the  three  groups  suggested  by  their  gross  cultural  and 
their  morphological  characteristics. 

First,  those  in  which  gemmation  of  spherical  or  oval  cells 
predominates,  having,  however,  an  associated  hyphal  form,  which 
may  bear  lateral  or  terminal  conidia,  and  which  may  undergo 
concatenate  hyphal  segmentation,  with  successive  abjunction 
of  terminal  oidium-like  cells.  The  formation  of  endospores  has 
not  been  observed  under  the  prescribed  conditions  of  Hansen. 
Organisms  i,  2,  5,  and  10  fall  into  this  group;  4,  6,  and  7  probably 
find  their  place  here,  but  the  observers  have  given  us  incomplete 
descriptions;    3,  in  its  cultural  life,  approximates  this  group. 

Second,  those  in  which  hyphal  forms  predominate,  prohferating 
as  indicated  above;  budding  is  always  found,  however,  but  lateral 
conidial  formation  may  be  sHght  or  entirely  absent;  endogenous 
spherules  form,  but  it  is  doubtful  that  they  are  spores.  True 
aerial  hyphae  do  not  form,  but  there  may  be  short  aerial  prickles. 
Organism  11  is  the  t>'pe;  3  probably  should  be  placed  here,  and 
also  9,  so  far  as  Gilchrist's  description  indicates;  the  last,  however, 
has  not  been  completely  worked  out. 

Third,  these  are  the  mould-fungi.  They  have  all  the  prohf- 
erative  possibilities  of  the  first  and  second  groups,  and  produce 
in  addition  aerial  hyphae,  which  bear  lateral  conidia  capable 
of  genmiation,  and  occasionally  form  terminal  ascospores,  or 
naked  spore  groups.  It  seems  quite  certain  that  the  cell  proto- 
plasm may  be  broken  up  into  large  numbers  of  spores,  but  com- 
plete proof  has  not  been  obtained.  Organisms  12,  13,  14,  15,  and 
16  constitute  the  group,  and  in  addition  17,  so  far  as  described. 
Buschke,  in  studying  Gilchrist's  organism  No.  3,  observed  that 
aerial  hyphae  formed  on  certain  media,  assigning  to  it  character- 
istics belonging  variously  to  hyphomycetes,  oidia,  and  blastomy- 
cetes. 

To  sum  up  the  question  of  reproduction:  All  organisms  repro- 
duce in  tissues  by  gemmation,  and  possibly  by  endogenous  spore 
formation,  the  latter  not  being  capable  of  proof  at  present;    in 


Oidiomycosis  of  the  Skin  and  Its  Fungi  159 

cultures,  by  gemmation,  lateral  conidium  formation,  and  mycelial 
spore  formation  following  the  oidium  t>'pe;  those  most  highly 
differentiated  by  the  production  of  conidium-bearing  aerial  hyphae, 
in  addition  to  all  the  other  methods.  There  is  thus  an  ascending 
scale  as  to  complexity,  the  lowest  represented  by  sac-like  budding 
cells,  the  highest  by  the  mould-fungi.  Endogenous  spore  forma- 
tion in  cultures  is  probable,  but  not  proved. 

The  various  organisms  produce  the  same  type  of  lesions  in 
animals,  but  there  are  clearly  different  degrees  of  virulence  among 
them.  (See  the  summary  of  literature  and  description  of  our 
cases.) 

classification  and  nomenclature. 

It  is  well  known  that  the  micro-organisms  of  the  simplest  con- 
struction, as  staphylococci,  are  capable  of  considerable  variations. 
Diphtheria  and  tubercle  bacilli  show  greater  variations  in  accord- 
ance with  their  more  complex  structure.  Brefield,  Hansen,  Jor- 
gensen,  and  others  cite  many  examples  of  variation  in  frutification 
and  morphology  among  the  genera  and  species  of  hyphomycetes. 
"Brefield  showed  that  many  Ustilagineae  (smut-fungi),  basidio- 
mycetes,  and  other  fungi  may  enter  upon  a  budding  fungus  stage." 

Sabouraud  fell  into  great  confusion  by  the  almost  numberless 
varieties  of  trichophytons  he  obtained  from  various  cases  of  ring- 
worm, which  were  identical  clinically. 

Hansen  has  showed  how  easy  it  is  to  produce  new  species  of 
yeasts.  From  an  isolated  cell  he  succeeded  in  obtaining  three 
distinct  species:  one  which  always,  one  which  rarely,  and  one 
which  never  formed  endogenous  spores. 

Jorgensen  states  that  a  certain  variety  of  Saccharomyces 
cerevisiae  grows  with  the  usual  ascus-like  cells  at  27°  C,  but  at 
75°  C.  grouped  colonies  with  mycelium-like  branches  are  obtained. 
What  is  considered  endogenous  spore  formation  in  the  saccharomy- 
cetes  is  retarded  or  accentuated  by  slight  variations  in  external 
conditions  (temperature,  age,  the  chemical  qualities  of  the  sur- 
rounding medium,  etc.). 

"The  majority  of  saccharomycetes  are  only  known  as  budding 
fungi  with  endogenous  spore  formation.     In  the  minority  a  mould 


i6o  Contributions  to  Medical  Science 

stage  is  also  known,  which  bears  a  certain  resemblance  to  dema- 
trium,  oidium,  or  moniha"  (Jorgensen). 

Among  the  lower  fungi  it  seems  that  the  higher  the  state  of 
organization  the  greater  the  possibilities  of  variations  in  mor- 
phology in  a  particular  genus  or  even  species. 

Continued  study  of  disease  processes  is  leading  to  the  conclusion 
that  a  particular  morbid  condition  is  associated  invariably  with  a 
particular  cause  or  a  group  of  causes.  We  are  drawing  closer 
constantly  to  the  ideal  of  specific  etiology. 

In  actinomycosis  and  trichophytosis,  however,  we  have  two 
examples  of  morbid  conditions  caused  by  groups  of  organisms,  the 
individuals  of  which  may  show  wide  morphological  variations. 
Those  who  have  studied  them,  however,  have  concluded  that  the 
members  of  each  group  are  very  closely  related,  and  consider  them 
as  members  of  a  common  genus  or  as  specific  adaptation  forms. 

It  is  of  the  greatest  importance  to  recognize  that  in  the  blasto- 
mycetic  dermatitis  of  Gilchrist,  twenty- three  cases  having  been 
given  careful  clinical  and  histological  study,  we  have  a  disease 
entity  which  is  uniform  clinically  and  histologically,  except  for 
unimportant  details.  It  has  been  separated  absolutely  from  some 
other  diseases  which  have  one  or  more  points  of  similarity  to  it. 

Of  equal  importance  is  the  fact  that  three  different  types  of 
organisms,  as  described  above,  appear  to  have  etiological  rela- 
tionship to  blastyomcetic  dermatitis.  A  priori,  one  would  conclude 
that  the  various  organisms  should  be  closely  related,  having  in 
mind  the  similar  conditions  in  actinomycosis  and  trichophytosis. 

The  study  of  cultures  shows  some  constant  differences  among 
the  organisms,  and,  on  the  other  hand,  many  more  common  points. 
All  may  proliferate  by  budding,  by  an  oidium-like  segmentation 
of  hyphae,  and  by  lateral  conidium  formation.  One  type  is  unique 
in  producing  aerial  hyphae,  another  is  conspicuously  oidium-like, 
and  the  third  blastomyces-like.  The  important  characteristics 
not  common  to  all,  so  far  as  we  know,  are  aerial  fructification  and 
fermenting  powers.  The  latter  is  absent  from  the  mould-fungi, 
and  from  four  of  the  blastomyces-like  organisms;  hence  this 
property  is  not  an  adequate  differential  point.  It  follows,  then, 
that  aerial  fructification  is  the  most  striking  differential  feature. 


Oidiomycosis  of  the  Skin  and  Its  Fungi  i6i 

Is  this,  then,  sufficient  to  disallow  classification  of  the  organisms 
under  a  common  genus  ? 

Having  consulted  the  works  of  De  Bary,  Curtis,  Cooke,  Under- 
wood, and  others,  I  find  that  there  are  not  greater  differences 
between  the  various  organisms  under  consideration  than  are 
commonly  allowed  to  certain  genera  of  the  hyphomycetes.  With 
this  as  a  basis,  and  considering  the  common  source  of  the  organisms, 
the  conclusion  is  reached  that  those  cultivated  from  the  various 
cases  of  blastomycetic  dermatitis  should  be  placed  in  a  common 
genus.  It  may  be  assumed  that  the  ability  to  form  aerial  hyphae 
has  in  some  cases  been  suppressed,  and  other  differences  are  explain- 
able along  the  same  line. 

Difficulty  arises  when  the  attempt  is  made  to  locate  this  genus 
in  a  botanical  system,  and  pathologists  cannot  hope  to  succeed 
where  botanists  have  failed.  In  concluding  that  they  are  hypho- 
mycetes, we  have  in  mind  the  statement  of  botanists  that  many 
hyphomycetes  are  merely  conidial  stages  of  certain  ascomycetes; 
yet  considering  the  most  highly  differentiated  (the  mould-fungi) 
as  the  appropriate  type  of  the  group,  the  character  of  the  fructifi- 
cation and  the  abundant  formation  of  hyphae  indicate  their  posi- 
tion under  hyphomycetes. 

Further  classification  can  be  accomplished  only  by  making 
comparisons  with  some  pre-existing  and  accepted  form.  In 
concluding  that  these  organisms  belong  to  the  oidia,  it  is  with 
the  knowledge  that  the  position  of  the  genus  is  uncertain,  and  that 
in  it  are  included  some  forms  which  are  said  to  represent  conidial 
stages  of  higher  organisms.  However,  among  the  organisms 
studied,  the  morphologic  characteristics  of  oidium  are  quite  con- 
stant, and  constitute  the  most  distinguishing  feature  of  the  group. 
The  fact  that  budding  takes  place  in  all  is  not  sufficient  justifica- 
tion for  considering  them  blastomycetes,  or  saccharomycetes,  as 
this  method  of  proliferation  is  likewise  found  in  the  conidia  of 
many  fungi,  particularly  the  oidium  forms;  it  seems  clear  that  the 
term  "  blastomyces "  is  not  sufficiently  broad. 

The  similarity  of  the  organisms  described  by  Busse,  Curtis,  and 
by  Ophiils  and  Moffitt  to  those  studied  in  this  article  suffices  to 
place  them  in  the  same  group  with  the  latter. 


1 62  Contributions  to  Medical  Science 

The  specific  names  applied  (see  Table  i,  pp.  148  f.)  represent 
peculiarities  of  the  organisms  as  far  as  possible. 

In  accordance  with  the  name  applied  to  the  organisms,  the 
infections  in  man  should  probably  be  spoken  of  as  oidiomycosis, 
and  the  disease  in  the  skin  as  oidiomycosis  of  the  skin. 

GENERAL  SUMMARY. 

It  has  been  generally  considered  that  thrush  is  the  only  example 
of  a  disease  caused  by  an  oidium,  the  process  being  limited  largely 
to  the  mucous  membranes  of  the  mouth,  pharynx,  and  esophagus. 
Observers  have  shown,  however,  that  the  parasite  of  thrush  may 
penetrate  to  the  submucosa  of  the  pharynx,  esophagus,  and 
stomach,  and  even  produce  thromboses  with  consequent  ulcera- 
tions. Of  greater  importance  are  the  observations  of  abscesses  in 
the  brain,  Uver,  and  kidney,  and  of  abscesses  and  areas  of  consoli- 
dation in  the  lungs,  the  conditions  being  associated  with  the 
presence  of  organisms  of  the  oidium  type.  In  the  most  of  such 
cases  there  was  a  concomitant  mucosal  thrush,  and  so  far  as  stated 
there  was  no  mixed  infection  in  the  abscesses. 

Also,  organisms  not  differing  essentially  from  Oidium  albicans 
have  been  cultivated  from  cases  of  tonsilitis,  thrush,  and  infections 
in  other  parts  of  the  body,  and  have  been  termed  saccharomycetes 
or  blastomycetes  by  their  observers. 

The  natural  pathogenicity  of  organisms  of  this  type  extends 
to  certain  animals,  notably  horses,  which  in  Japan,  Italy,  Russia, 
and  some  other  countries  suffer  epidemically  from  a  stubborn 
burrowing  infection  of  the  skin,  superficial  lymphatics,  and  upper 
respiratory  passages.  Fermi  and  Aruch,  and  Tokishige  have 
cultivated  "blastomycetes"  from  the  lesions  of  such  cases,  and 
proved  the  specificity  of  the  organisms  for  the  disease.  Guinea- 
pigs  also  appear  to  suffer  from  invasion  by  blastomycetoid  organ- 
isms. 

Aside  from  the  theoretical  relationship  of  yeast-fungi  to  malig- 
nant tumors,  the  current  impetus  to  their  study  originated  in 
Busse's  discovery  of  a  generalized  fatal  case  of  "  Saccharomycosis 
hominis"  in  1894.  Curtis  followed  with  a  somewhat  similar  case. 
Gilchrist  in   1896  greatly  increased   the  general  interest  in   the 


Oidiomycosis  of  the  Skin  and  Its  Fungi  i6 


o 


subject  by  the  description  of  his  second  case  of  ''blastomycetic 
dermatitis"  and  the  organism  cultivated  from  it.  Since  that 
time  the  number  of  reported  cases  of  the  infection  in  the  skin  has 
increased  to  twenty-six.  Of  this  number  fifteen  have  been  dis- 
covered in  Rush  Medical  College,  the  present  report  including 
twelve  cases.  (As  noted,  some  of  these  cases  have  appeared  in 
the  literature  previously.)  Clinical  study  was  possible  in  ten  of 
the  cases,  the  other  two  being  museum  specimens  which  were  origi- 
nally considered  tuberculous.  All  were  given  thorough  histological 
study.  It  was  possible  to  attempt  the  cultivation  of  organisms 
from  nine  out  of  the  twelve  cases.  Only  seven  permanent  cultures 
were  obtained,  however,  the  eighth  dying  before  it  could  be  studied 
and  another  case  yielding  no  oidium  or  yeast-like  organism. 

There  is,  essentially,  a  uniform  clinical  history,  which  is  briefly 
as  follows: 

1.  An  incipient  papule  which  becomes  pustular,  yielding  a 
glairy,  somewhat  tenacious  pus. 

2.  Gradual  extension  of  the  ulcerating  surface,  which  is  soon 
covered  with  coarse,  soft,  and  friable  papillae,  and  is  surrounded 
by  a  reddened  areola  in  which  many  minute  abscesses  are  visible. 

3.  Cicatricial  healing  in  the  oldest  portions  of  the  lesion  as  the 
border  advances. 

4.  A  variable  amount  of  pain,  depending  on  the  site  involved, 
upon  a  temporary  increase  or  decrease  of  the  virulence  of  the 
specific  infection,  and  the  amount  of  secondary  infection  present. 

5.  The  absence  of  lymphatic  glandular  involvement  in  a  great 
majority  of  cases.  (It  seems  probable,  however,  when  generalized 
infection  occurs,  that  it  may  take  place  through  the  medium  of 
the  lymphatic  system.) 

6.  The  protracted  and  progressive  course  of  the  disease,  which 
may  extend  over  ten  or  twelve  years,  and  in  the  face  may  cause 
great  cicatricial  deformity. 

7.  Periods  of  rapid  extension,  interrupted  by  periods  of  relative 
quietude. 

8.  The  tendency  of  the  scar  to  eventually  approach  the  appear- 
ance of  normal  skin. 

9.  The  upper  extremities  (hand  and  fore-arm)  and  the  face  are 


164  Contributions  to  Medical  Science 

most  frequently  attacked,  although  no  portion  of  the  skin  is  proof 
against  invasion. 

10.  Extension  to  mucous  surfaces  does  not  occur  readily. 

11.  The  absence  of  general  toxic  disturbances,  attributable 
to  the  local  infection. 

The  gross  anatomy  is  characteristic  and  uniform  in  all  essen- 
tials. In  addition  to  the  features  mentioned  in  i,  2,  and  3,  above, 
naked-eye  inspection  of  a  cross-section  shows,  from  without 
inward : 

1.  A  papillary  zone,  composed  of  a  superficial  layer  of  isolated 
villiform  processes,  and  a  deeper  layer  of  similar  processes  which 
are  united  side  by  side. 

2.  A  homogeneous,  vascularized,  grayish-red,  cellular  zone, 
in  which  are  formed  minute  abscesses. 

3.  An  unaltered  layer  of  subcutaneous  fat,  as  the  limit  of  deep 
extension. 

A  positive  diagnosis  is  readily  made  by  microscopic  demonstra- 
tion of  the  organisms  in  the  pus  of  the  miHary  abscesses,  or  in  the 
verrucous  processes  as  described. 

Stained  sections  of  the  tissue  exhibit  the  following  histological 
features:  (i)  a  vast  amount  of  " carcinomatoid "  epithehal 
hyperplasia;  (2)  minute  intra-epithelial  abscesses;  (3)  a  granulo- 
matous condition  in  the  corium,  characterized  by  masses  of  plasma 
cells,  minute  abscesses,  and  tuberculoid  nodules  and  giant  cells; 
(4)  the  presence  of  the  spherical,  capsulated,  budding  organism, 
particularly  in  the  epidermal  and  subepidermal  abscesses,  but  also 
distributed  unevenly  and  in  small  numbers  in  epithelial  masses  and 
granulation  tissue. 

The  study  of  plasma  cells  as  seen  in  the  skin  infection  indicates 
that  they  play  some  part  in  the  production  of  new  fibrous  tissue; 
not,  however,  being  transformed  into  connective-tissue  cells, 
but  probably  providing  some  substance  which  is  utilized  by  these 
cells  in  the  process  of  fiber  formation.  The  fate  of  the  plasma  cell 
in  this  relationship  is  gradual  disintegration,  and  the  result  accom- 
plished is  a  peripheral  zone  of  new  fibrous  tissue  as  an  obstruction 
to  extension  of  the  infection. 

Mast-cells   proliferate   extensively,    and   morphologically   and 


Oidiomycosis  of  the  Skin  and  Its  Fungi  165 

tinctorially  are  of  four  types:  (i)  leukocytic;  (2)  connective- 
tissue  cell  type;  (3)  the  plasma-mast  cell;  (4)  the  mast-cell  with 
a  halo.  Their  fate  and  function  have  not  been  made  out,  but 
it  is  not  clear  that  they  are  " mucinoblasts "  (Harris);  their 
anatomical  distribution  speaks  against  this  theory,  and  the  tinc- 
torial test  is  not  conclusive. 

It  has  been  made  out  that  in  many  cases  the  multinuclear 
giant  cells  seen  in  the  epidermis  and  intra-epithelial  abscesses  are 
of  epithelial  origin. 

Those  cases  from  which  a  mould-fungus  has  been  isolated 
distinguish  themselves  from  all  others  by  the  presence  of  great 
numbers  of  eosinophils  in  all  parts  of  the  diseased  tissue. 

The  organism  in  human  tissue  has  an  average  size  of  about  ten 
microns  and  never  forms  threads  similar  to  those  seen  in  cultures. 
Proliferation  (in  tissue)  is  by  gemmation;  it  seems  probable,  also, 
that  endogenous  spores  may  form. 

In  addition  to  blastomycetic  dermatitis,  a  number  of  cases 
of  a  generalized  infection  with  concomitant  cutaneous  lesions  have 
been  described  (particularly  by  Rixford  and  Gilchrist),  the  disease 
having  a  close  clinical  similarity  to  diffuse,  ulcerative,  and  miliary 
tuberculosis.  Rixford  and  Gilchrist,  and  others,  have  considered 
the  infection  protozoic  in  nature,  because  of  the  structure  of  the 
large,  spherical,  capsulated  organisms,  and  their  inability  to  grow 
them  on  artificial  media.  From  a  case  of  this  type,  Ophiils  and 
Mofl&tt  recently  cultivated  a  mould-fungus  pathogenic  for  animals. 
This  mould-fungus  appears  identical,  morphologically  and  patho- 
genetically,  with  that  cultivated  from  five  cases  of  this  series. 
The  cutaneous  lesions  in  the  so-called  protozoic  disease,  moreover, 
are  identical  in  gross  and  histological  appearances,  with  those 
of  oidial  dermatitis,  all  the  evidence  pointing  to  a  close  relationship 
between  the  two  conditions. 

The  organisms  which  have  been  cultivated  by  various  observers 
from  oidiomycosis  of  the  skin  fall  into  three  groups:  (i)  a  blasto- 
mycetoid;  (2)  an  oidium-like;  and  (3)  a  hyphomycetoid  group. 
All  the  organisms  exist  in  tissue  only  in  the  blastomycetoid  form. 
On  culture  media  the  first  group  exists  chiefly  as  spherical  or  oval 
budding  cells,  but  under  suitable  conditions  may  produce  abundant 


1 66  Contributions  to  Medical  Science 

mycelium.  The  second  forms  submerged  myceKum  which  breaks 
up  into  chains  of  "spores";  while  proliferation  by  budding  is  not 
a  prominent  feature.  The  third  produces  fruit-bearing  aerial 
h>'phae,  submerged  hyphae  similar  to  those  of  the  first  two  groups 
and  is  also  capable  of  multiplying  by  gemmation.  Hence  from  the 
first  to  the  third  group  there  is  a  gradual  transit.  The  specific 
characteristics  of  individual  organisms  remain,  for  the  most  part, 
constant,  so  it  seems  that  an  organism  belonging  to  one  group  is 
not  readily  induced  to  grow  Uke  those  of  the  group  higher  or  lower. 
Sufficient  variations,  however,  have  been  noted,  to  point  to  the 
possibility  of  a  lower  organism  acquiring  some  of  the  properties 
of  a  higher,  or  vice  versa.  (Quite  recently  the  mould-fungus  from 
Case  VIII  began  to  grow  with  the  peculiar  folded  surface  produced 
by  the  organism  from  Case  III,  losing  its  mouldy  surface  at  such 
places.)  It  seems  probable  that  those  organisms  which  grow 
chiefly  as  budding  fungi  or  in  the  form  of  segmented  threads  are 
examples  of  a  reversion,  on  the  part  of  a  higher  organism,  to  a 
more  simple  or  continued  conidial  form.  The  organisms,  further- 
more, fall  into  two  physiological  divisions:  the  fermenting,  includ- 
ing the  first  and  second  groups;  and  the  non-fermenting,  the 
third  group.  The  morphological  and  biological  limitations  assigned 
to  oidium  by  botanists  indicate  the  placing  of  all  the  organisms 
cultivated  from  cases  of  "  blastomycetic  dermatitis,"  Saccharomy- 
cosis  hominis,  and  probably  the  so-called  protozoic  disease  in  this 
as  a  common  genus,  the  different  species  being  well  differentiated. 
The  pathogenicity  of  the  different  organisms  for  animals  varies. 
Subcutaneous  abscesses,  septicemia,  pulmonary  consolidation, 
and  pyemic  foci  have  been  induced  by  various  methods  of  inocula- 
tion. Attempts  to  reproduce  a  typical  lesion  in  man  by  inocula- 
tion of  pure  cultures  have,  so  far,  failed.  Since  susceptibility 
to  the  infection  seems  to  be  quite  rare,  these  few  failures  to  repro- 
duce the  disease  in  man  obviously  cannot  be  regarded  as  definitely 
settling  the  question.  On  the  other  hand,  the  claim  that  these 
organisms  are  specific  for  the  disease  is  not  out  of  range  of  strict, 
technical  criticism,  until  the  lesions  seen  in  man  are  reproduced 
by  the  inoculation  of  pure  cultures  into  animals  or  man.     Aside 


Oidiomycosis  of  the  Skin  and  Its  Fungi  167 

from  this  crucial  test,  the  etiological  position  of  the  organisms 
seems  unquestionable. 

Until  the  distinguishing  features  of  the  disease  are  grasped, 
it  is  natural  to  expect  adverse  and  biased  criticisms,  a  noteworthy 
example  of  which  appeared  recently.^ 


conclusions. 

1.  The  so-called  protozoic  disease  of  Posadas,  Wernicke,  and 
others;  Busse's  and  Curtis'  Saccharomycosis  hominis;  and  Gil- 
christ's blastomycetic  dermatitis  are  various  manifestations  of 
the  same  disease. 

2.  The  condition  in  the  skin  possesses  constant  clinical  and 
histological  characteristics,  which  separate  it  positively  from  all 
other  skin  diseases,  particularly  verrucous  tuberculosis,  carcinoma, 
and  syphilis. 

3.  The  organisms  isolated  from  various  cases  differ  in  minor 
respects  among  themselves,  but  are  so  closely  related  morphologi- 
cally and  biologically  as  to  justify  their  inclusion  in  a  common 
genus,  oidium;  they  are  thus  analogous  in  a  pathogenetic  sense  to 
the  fungi  which  cause  actinomycosis,  and  to  those  causing  tricho- 
phytosis. 

4.  The  variations  among  the  organisms  allow  the  recognition 
of  three  morphological  types:  (i)  blastomycetoid  or  yeast-like;  (2) 
oidium-Hke;    (3)  hyphomycetoid. 

5.  There  are  two  histological  forms  of  the  disease  in  the  skin, 
the  eosinophilous  and  the  non-eosinophilous,  the  former  being 
associated  with  the  mould  type  of  the  organism. 

6.  In  accordance  with  conclusion  3,  oidiomycosis  is  an  appro- 
priate term  for  the  conditions  caused  by  the  organisms,  and 
Oidiomycosis  cutis  for  the  disease  as  it  occurs  in  the  skin. 

7.  Aside  from  the  infections  considered  in  this  communication, 
certain  cases  which  have  been  described  in  the  literature  from 
time  to  time  indicate  that  oidium-Hke  organisms  may  cause  other 
severe  pathological  conditions  in  man. 

'  Kromayer,  Ernst,  Centralbl.  filr  allg.  Path,  und  path.  Anat.,  igoo,  ii,  p.  777. 


1 68  Contributions  to  Medical  Science 


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Zenker,  F.  A.    Jahresb.  der  Gesellsch.  f.  Natur  u.  Heilkunde  in  Dresden,   1860-61,  51 


EXPLANATION  OF  PLATES. 

Plate  i,  Fig.  la. — Photograph  of  lesion  in  Case  VII. 
Fig.  16. — Photograph  of  lesion  in  Case  IX. 

Plate  2,  Fig.  ic. — Case  V.  A  budding  " blastomyces "  and  a  mature  single 
form  in  an  intra-epithelial  abscess.  Hematoxylin  and  eosin.  Photomicrograph 
X 1 200. 


174  Contributions  to  Medical  Science 

Fig.  2. — Case  III.  A  giant  cell  of  probable  epithelial  origin,  containing  a  pair 
of  degenerating  organisms.    Hematoxylin  and  eosin.     Photomicrograph  X 1200. 

Plate  3,  Fig.  3. — Case  VII.  A  pair  of  organisms  in  an  incipient  intra-epithe- 
lial  abscess;  also  a  single  mature  form  between  epithelial  cells.  Hematoxylin  and 
eosin.     Photomicrograph  X1200. 

Fig.  4. — Case  HI.  Three  organisms  in  an  atypical  chain,  lying  in  an  abscess  of 
corium.     Polychrome  methylene  blue.     Photomicrograph  X1200. 

Plate  4,  Fig.  5. — Case  III.  Hanging-drop  culture.  Tortuous,  segmented 
hyphae;  an  oidial  form.  Photomicrograph  g  in.  objective,  ocular  No.  4.  Bausch  and 
Lomb. 

Fig.  6. — Margin  of  colony  in  Fig.  5.     X1200. 

Fig.  7. — Case  VII.  Organism  in  plate  culture.  The  feathery  nature  of  colonies 
is  shown  well.     Photograph  slightly  reduced. 

Plate  5,  Fig.  8. — Photomicrograph  of  colony  shown  in  Fig.  7.  Branching 
hyphae  giving  off  imicellular  shoots. 

Plate  6,  Fig.  9. — Case  HI.  Single  plate  colony  of  organism.  Photomicro- 
graph X60. 

Fig.  10. — Case  HI.  Higher  magnification  of  colony.  Hyphae  are  segmented, 
and  older  cells  contain  refractive  spore-like  bodies.     Photomicrograph  X120. 

Plate  7,  Fig.  ii. — Case  \TI.  Submerged  hypha,  segmented,  and  sprouting 
spherical  and  elongated  conidia;  spherical  cells,  6-8  microns  in  diameter;  elongated 
cells,  about  4  microns  thick.  Each  segment  contains  from  two  to  four  small  refractive 
bodies. 

Fig.  12. — Case  VII.  Forms  seen  in  a  nodule  in  the  lung  of  a  dog  which  died 
from  an  intravenous  inoculation.  Drawn  from  a  fresh  mount  in  water.  A,  B,  and 
D  measured  8  10,  and  12  microns  respectively;  C  and  E,  15  microns  each,  enlarged. 
The  refractive  bodies  in  A,  B,  and  D  are  similar  to  those  in  C.  F,  spherical  spore- 
like bodies  l3dng  free  among  and  sometimes  included  in  tissue  cells;  from  i  to  3  or  4 
microns  in  diameter.     E,  a  budding  organism  with  an  adventitious  capsule. 

Fig.  13. — Case  I.  Drawing  from  a  drop  mount  of  a  bouillon  culture.  Hyphae 
with  terminal  and  lateral  conidium  groups.  Cells  contain  the  nucleus-like  structures 
spoken  of.     X 1200  (about). 

Fig.  14. — Case  I.  Sprouting  of  hyphae  in  blood-serum  of  dog,  showing  nucleus- 
like bodies.     X 1 200. 

Plate  8,  Fig.  15. — Case  I.  Forms  seen  in  the  pus  produced  by  a  subcuta- 
neous inoculation  in  guinea-pig. 

Fig.  16. — Case  I.  Hanging-drop  culture  in  which  the  oidium  morphology  is 
maintained  somewhat  imperfectly.     Photomicrograph  X 1 200. 

Plate  9,  Fig.  17. — Case  VII.  Terminal  spore-groups.  Liquid  culture. 
10-12  microns. 

Fig.  i8a. — Case  HI.  Plasmolysis,  pseudo-spore  formation  or  perhaps  true  spore 
formation,  occurring  in  segmented  hyphae  fragments.  Width  about  8  microns; 
enlarged. 

Fig.  18&. — Case  HI.  The  morphology  suggests  endospores,  but  the  bodies 
appear  structureless.    The  cell  measured  30  microns  in  diameter. 


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Oidiomycosis  of  the  Skin  and  Its  Fungi  175 

Plate  10,'  Fig.  i. — A  typical  low-power  view  of  a  skin  lesion,  showing  the 
carcinomatoid  epithelial  hyperplasia  and  intra-epithelial  abscesses.  From  Case  III. 
Methylene  blue.     Bausch  and  Lomb  \  in.  objective,  No.  2  eyepiece. 

Fig.  2. — An  intra-epithelial  abscess,  containing  a  group  of  organisms.  The 
green  color  reaction  of  the  capsules  is  perhaps  somewhat  exceptional.  From  Case  IV. 
Polychrome  methylene  blue.     Bausch  and  Lomb  j'^  in.  oil  objective,  eyepiece  No.  2 

Fig.  3. — Illustrating  the  "cobble-stone  appearance"  of  the  rete  mucosum,  fre- 
quently seen.  The  chromophilic  substance  of  the  cell  body  is  concentrated  in  a 
peripheral  segment  of  the  cells  in  considerable  areas  of  tissue,  evidently  an  example 
of  chromatolysis.  From  a  laboratory  case.  Polychrome  methylene  blue.  Bausch  and 
Lomb  i^a  in.  oil  objective,  eyepiece  No.  2. 

Fig.  4. — Mycotic  focus  in  kidney  of  a  mouse  which  died  from  subcutaneous 
injection  of  the  organism  from  Case  I.  The  organisms  within  tubules  and  in  inter- 
stitial tissue.  Small  and  streptothrix-like  forms.  Granular  degeneration  of  tubular 
cells.     Polychrome  methylene  blue.     Same  magnification  as  Fig.  3. 

Plate  ii,'  Fig.  5. — Showing  a  change  that  occurs  in  the  prickles  of  the  mucosal 
cells  in  the  vicinity  of  an  abscess.  The  processes  apparently  are  transformed  into 
a  semi-comified  capsule  for  the  cell. 

Figs.  8  and  6  show,  the  former  a  very  early,  the  latter  a  later,  stage,  in  the  process 
of  epithelial  multinuclear  giant  cell  formation. 

Fig.  7. — A  small  intra-epithelial  abscess,  surrounded  by  a  partially  cornified 
capsule.    A  pair  of  organisms  and  two  eosinophile  leukocytes  are  seen  in  the  abscesses. 

Figs.  5-8. — Hematoxylin  and  eosin.  Bausch  and  Lomb  iV  in.  oil  objective, 
eyepiece  No.  2. 

Plate  12,'  Fig.  9. — Cells  seen  in  the  lung  of  a  dog  which  died  from  an  intra- 
venous inoculation  of  the  organism  cultivated  from  Case  VIII,  stained  with  hema- 
toxylin, a)  A  large  organism,  the  protoplasm  of  which  contains  many  deeply  staining 
spherical  bodies  (spores?),  b)  An  alveolar  cell  containing  a  single  organism,  c)  A 
mass  of  organisms  surrounded  by  a  finely  granular  substance.  They  lay  within  an 
air  sac,  dilating  it.  d)  A  multinuclear  giant  cell  (of  epithelial  origin  ?)  containing 
many  organisms.     Bausch  and  Lomb  objective  iV  in.,  eyepiece  No.  2. 

Fig.  id. — The  relation  of  plasma  cells  to  new  forming  fibrous  tissue,  as  shown  by 
a  carbol-fuchsin  and  water-blue  stain.  From  Case  I.  This  figure  and  the  one  fol- 
lowing much  enlarged  from  observations  with  iV  in.  oil  immersion  objective. 

Fig.  II. — The  occurrence  of  hyalin  spherules  in  plasma  cells.  Case  I.  Carbol- 
fuchsin  and  water-blue. 

■  Colored  plates  in  the  original. 


AN   ORGANISM  FROM   CUTANEOUS  OIDIOMYCOSIS 
(BLASTOMYCOSIS).^ 

REMARKS   ON   CLASSIFICATION. 

H.    T.     RiCKETTS. 
{From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

I  desire  to  record  a  hitherto  unreported  case  of  blastomycosis, 
or  oidiomycosis  of  the  skin,  from  which  the  fungus  was  obtained 
in  pure  culture. 

I  saw  the  patient  at  the  request  of  Dr.  Porter  of  Champaign, 
111.,  in  his  office  in  the  fall  of  1902.  It  was  the  plan  to  publish  a 
full  clinical  and  pathologic  report  of  the  case  at  the  time,  but  the 
patient  refused  to  be  photographed  or  allow  the  removal  of  tissue 
for  study.  A  report  on  the  organism  has  been  delayed  with  the 
expectation  that  other  material  might  come  to  hand  which  could 
be  incorporated. 

The  disease  was  of  several  years'  duration,  had  its  site  on  the 
left  of  the  face,  had  involved  the  lower  eyelid,  and  presented  the 
verrucous  surface  and  miliary  abscesses  characteristic  of  the  pro- 
cess. Fresh  preparations  of  the  contents  of  miliary  abscesses 
revealed  numerous  spherical,  capsulated,  budding  cells. 

It  was  possible  to  obtain  a  few  plain  agar  culture  tubes  from  the 
laboratory  of  the  state  university;  these  were  inoculated  with 
the  pus  of  the  small  abscesses,  which  are  almost  invariably  found 
in  or  beneath  the  unbroken  epidermis  which  surrounds  the  ulcerated 
or  verrucous  area.  No  incubator  was  at  hand,  but  the  summer 
temperature  was  favorable  to  the  development  of  micro-organisms. 
At  the  end  of  twenty-four  hours  a  few  minute  colonies  had  developed 
on  one  tube;  these  proved  later  to  be  staphylococci.  On  the 
third  day  small  grayish  points,  which  were  less  conspicuous  than 
the  colonies  first  noted,  were  seen  on  all  tubes.  These  developed 
slowly,  and  in  a  few  days  formed  opaque  circular  areas  on  the 
surface  of  the  medium,  the  center  of  each  colony  became  marked  by 
a  blunt,  elevated  point,  and  a  little  later  the  growth  extended  into 
the  substratum.     In  about  ten  days  it  showed  a  downy  surface, 

"  From  Trans.  Chic.  Path.  Soc,  igo4,  6.  p.  113. 

176 


An  Organism  from  Cutaneous  Oidiomycosis  177 

a  feature  which  became  more  pronounced  during  subsequent 
days,  and  eventually  resulted  in  the  marked  mouldy  appearance 
which  is  more  or  less  characteristic  for  this  type  of  the  organism. 

The  growth  in  the  substratum  of  the  medium  is  made  up  of 
hyphae,  which  are  divided  into  segments  of  varying  lengths,  and 
which  give  oflf  abundant  branches.  It  is  not  unusual  to  find  the 
segments  of  a  single  thread  separated;  this  is  interpreted  as  pro-  ' 
liferation  by  segmentation.  The  aerial  threads  are  finer,  more 
compact  in  appearance,  and  are  not  so  prominently  segmented 
as  those  of  the  underlying  growth.  They  have  many  long  fine 
branches,  and  in  addition  shorter  ones,  the  terminal  extremities 
of  which  possess  either  a  single  small  spherical  cell,  or  a  group  of 
such  cells.  These  are  the  conidia  mentioned  in  previous  descrip- 
tions. 

In  bouillon,  growth  occurs  in  the  form  of  coherent,  delicate 
tufts,  which  may  reach  large  proportions  in  the  course  of  two  or 
three  weeks. 

The  organism  was  not  pathogenic  for  guinea-pigs  in  subcuta- 
neous inoculations. 

In  the  particulars  enumerated  and  in  all  others,  this  culture 
appears  to  be  identical  with  that  of  an  organism  which  I  previously 
cultivated  from  five  cases  of  cutaneous  blastomycosis,  and  first 
described  in  a  paper  entitled  **A  New  Mould-Fungus  as  the  Cause 
of  Three  Cases  of  So-called  Blastomycosis  or  Oidiomycosis  of  the 
Skin."^  A  consideration  of  the  whole  subject,  including  a  descrip- 
tion of  the  organisms  cultivated  by  various  investigators,  was 
given  later  in  the  Journal  of  Medical  Research  (1901,  6,  p.  374)- 

The  characteristics  of  the  organisms  described  in  literature 
and  of  those  which  came  under  my  own  observation  were  such 
that,  in  the  latter  paper,  they  were  divided  into  three  groups. 
This  division  was  based  on  both  the  cultural  and  the  microscopic 
appearances  of  the  three  types.  In  the  first  group  are  those  organ- 
isms which,  on  agar  slants,  form  a  smooth  pasty  growth,  without 
aerial  hyphae,  and  which  may  or  may  not  penetrate  the  sub- 
stratum. Proliferation  is  chiefly  by  a  budding  process,  but  often 
myceHal  formation  and  myceHal  segmentation  are  conspicuous. 

The  second  group  is  intended  to  include  those  organisms  which 

'  Jour.  Boston  Sac.  Med.  Sc,  1909,  s,  p.  453. 


178  Contributions  to  Medical  Science 

grow  with  a  granular  surface,  which  later  may  present  rugae  and 
folds,  but  do  not  develop  true  aerial  hyphae;  minute  short  spikes 
may  project  slightly  above  the  surface.  The  depth  of  the  medium 
is  invaded.  Proliferation  in  this  type  is  chiefly  by  the  formation 
of  a  segmented  mycelium,  the  elements  of  which  eventually  separate 
after  the  manner  of  oidium. 

The  third  group  consists  of  the  mould-fungi  which  have  been 
cultivated  from  the  disease.  These  prohferate  by  the  methods 
enumerated  for  the  first  two  groups,  and  in  addition  conidia  are 
formed  on  the  aerial  hyphae. 

The  appearances  on  other  media,  especially  bouillon,  also  show 
features  which  are  peculiar  to  each  group. 

In  spite  of  cultural,  morphologic,  and  certain  biologic  differ- 
ences which  are  noted  among  the  three  types,  other  features  which 
they  possess  in  common,  and  the  fact  that  the  three  are  found  in 
the  same  disease,  indicate  that  they  are  closely  related  organisms. 
For  this  reason  they  were  all  considered  to  occupy  the  same  generic 
position,  which,  as  nearly  as  could  be  determined,  was  that  of 
oidium.  The  fact  that  all  the  organisms  may  prohferate  by  a 
budding  process,  and  in  tissues  do  so  constantly,  is  no  argument 
against  referring  them  to  the  genus  oidium,  inasmuch  as  the  oidia 
and  even  other  h^^^homycetes  are  known  to  lapse  into  a  budding 
stage. 

Holding  these  views,  it  has  been  interesting  to  note  certain 
cultural  changes  which  indicate  the  close  relationship  of  the  three 
types.  I  refer  especially  to  the  fact  that  an  organism  belonging 
to  the  first  group,  one  which  habitually  grows  with  a  smooth 
pasty  surface,  has  been  seen  to  grow  with  the  uneven  and  rugated 
surface  characteristic  of  group  2 ;  and  that  every  one  of  the  mould- 
fungi  which  I  have  cultivated  has  at  one  time  or  another  grown 
without  the  moulded  surface,  also  resulting  in  an  appearance  which 
approaches  that  of  group  2.  Nevertheless,  certain  constant 
differences  and  the  tendency  to  return  to  the  original  form  of 
growth  preclude  the  possibility  of  considering  them  identical. 

It  is  of  interest  that  nearly  all  the  cultures  which  have  been 
reported  in  the  past  three  years  are  of  the  mould  t>TDe.  Hence  it 
seems  probable  that  the  mould-fungus  will  prove  to  be  the  pre- 
dominating organism  in  oidiomycosis. 


THE  IMMUNOLOGICAL  REACTIONS  OF  OIDIOMYCOSIS 
(BLASTOMYCOSIS)  IN  THE  GUINEA-PIG.' 

Benjamin   Franklin   Davis. 

{From  the  Department  of  Pathology  and  Bacteriology,  the  University  of  Chicago,  Chicago,  III.) 

The  object  of  this  work  was  to  examine  the  mode  of  resistance 
of  guinea-pigs  to  oidiomycetic  infection;  to  determine  whether  it 
might  be  possible  to  increase  this  resistance  by  immunization, 
and,  if  so,  to  investigate  the  factors  upon  which  this  increase  in 
resisting  power  depended. 

LITERATURE. 

For  a  full  discussion  of  the  relation  of  oidioid  and  blastomy- 
cetoid  organisms  to  human  and  animal  diseases  and  for  literature 
as  to  their  relative  pathogenicity  for  various  animals,  the  reader 
is  referred  to  the  work  of  Ricketts,"  Brown,^^  Hektoen,^^  Mont- 
gomery and  Ormsby,^^  and  Spiethoff.''  The  papers  considered 
here  are  those  which  have  dealt  somewhat  specifically  with  the 
question  of  the  mode  of  resistance  of  animals  to  infection  with 
mould-fungi,  and  with  the  demonstration  of  antibodies  in  the 
infected  animals. 

^Metchnikoflf'  (1884)  demonstrated  that  phagocytosis  is  the  chief  means  of  defense 
of  the  daphnia  against  infection  with  a  mould-fungus  christened  by  him  "Monospora 
bicuspidata."  This  was  followed  by  a  number  of  articles  by  Ribbert^  (1887)  who 
was  inclined  to  believe  that  mould-fungi  (Schimmelpilze)  and  pathogenic  schizomycetes 
injected  into  rabbits  are  ingested  by  leukocytes  within  which  they  undergo  intra- 
cellular digestion.  Charrin  and  Ostrowsky*  (1896)  observed  that  immunized  animals 
were  but  moderately  resistant  to  reinfection  with  Oidium  albicans.  Roger'  (1896) 
obtained  considerable  resistance  to  Oidium  albicans  by  vaccination.  Rabbits  given 
repeated  intravenous  injections  of  sublethal  doses  became  able  to  resist  infection 
by  double  the  lethal  dose.  In  normal  serum  the  organisms  grew  quite  readily;  in 
immune  serum  they  were  first  agglutinated,  then  became  hyaline,  the  capsule  disin- 
tegrated, and  later  attempts  to  obtain  cultures  showed  that  the  oldia  were  dead. 
Schattenfroh'  (1896),  using  a  non-pathogenic  yeast,  found  that  the  sera  of  animals 
were  not  bactericidal  but  that  peritoneal  exudates  were  markedly  so.  He  concluded 
that  the  germicidal  power  of  peritoneal  exudates  depends  on  phagocytosis.  Gilkinet^ 
(1897)  believed  from  the  results  of  his  experiments,  (i)  that  beer  yeasts  (Saccharomyces 
cerevisiae)  introduced  into  rabbits  either  intravenously  or  subcutaneously  produce 
» From  Jour.  Inject.  Dis.,  1911,  8,  p.  190. 

179 


i8o  Contributions  to  Medical  Science 

neither  local  nor  general  symptoms;  (2)  that  such  yeasts  do  not  multiply  in  living 
tissues;  (3)  that  they  are  destroyed  within  a  very  short  time,  and  cannot  be  discovered 
in  the  bodies  of  the  animals  by  any  method;  (4)  that  this  destruction  is  brought  about 
by  the  substances  in  the  plasma  and  is  not  a  function  of  the  body  temperature,  of 
chemical  reaction,  or  of  the  absence  of  nutritive  substances,  but  is  a  specific,  unknown 
property  of  the  organic  fluids;  (5)  that  this  destructive  property  is  not  dependent 
upon  the  formed  elements  of  the  blood,  but  is  exercised  in  the  same  degree  by  all  the 
body  liquids;  and,  finally,  (6)  that  this  property  is  itself  destroyed  by  heating  at  55°  C. 
He  found  that  yeast  mixed  with  ox  serum  or  rabbit  serum  and  kept  at  36°  C.  for  from 
two  to  four  days  lost  its  power  of  growth  on  suitable  media,  but  that  if  such  mixtures 
were  kept  at  room  temperature  for  corresponding  periods,  the  yeasts  were  unharmed. 
He  placed  porous  tubes  containing  blastomycetes  in  the  peritoneal  cavities  of  three 
rabbits.  The  first  rabbit  was  examined  after  four  days;  the  yeast  cells  were  not 
much  changed,  and  were  not  in  contact  with  leukocytes;  in  the  second  rabbit,  killed 
after  nine  days,  and  in  the  third  rabbit,  killed  after  twelve  days,  there  were  but  few 
normal  yeast  forms  and  apparently  all  were  dead,  since  cultures  were  negative. 
Leukocytes  were  not  in  contact  with  the  organism.  The  author  produced  edema  of 
the  rabbit's  leg  by  tight  bandaging;  such  edematous  fluid,  free  from  leukocytes,  killed 
yeasts  in  three  days  at  37°  C.  Jona*  (1897)  gave  rabbits  intraperitoneal,  intravenous, 
and  subcutaneous  injections  of  a  non-pathogenic  yeast,  Saccharomyces  apiculatus. 
He  concluded  that  in  all  cases  the  organisms  were  destroyed  within  a  few  hours  through 
the  influence  of  the  body  fluids.  Organisms  injected  into  the  peritoneal  cavity,  or 
subcutaneously,  did  not  make  their  way  into  the  blood.  Obici'  (1898)  found  that 
after  repeated  injections  of  the  toxin  (filtered  broth  culture)  and  small  quantities  of  the 
spores,  an  immunity  was  established  in  rabbits,  against  Aspergillus  fumigatus.  This 
immunity,  however,  was  incomplete,  and  vanished  in  a  short  time.  He  did  not 
believe,  with  Ribbert,  that  phagocytosis  was  of  such  paramount  importance  in  the 
defense  of  the  animal  body  against  this  infection,  as  he  saw  evidences  of  degeneration 
in  organisms  in  the  absence  of  phagocytic  cells.  Skchiwan'"  (1899)  experimented 
on  guinea-pigs  and  rabbits  with  Saccharomyces  tumefaciens  of  Curtis.  He  con- 
cluded that  the  body  fluids  are  not  bactericidal  since  the  organisms  protected  from 
the  leukocytes  by  celloidin  sacs  grew  well  on  artificial  media  after  being  in  the  peri- 
toneal cavity  of  the  animal  for  four  days.  On  the  other  hand,  yeasts  injected  directly 
into  the  peritoneal  cavity  were  phagocyted  by  certain  cells  and  digested.  The  pha- 
gocyted  yeasts  lost  their  staining  power  in  from  two  to  four  days,  as  well  as  their 
power  to  grow  when  planted  on  suitable  media.  The  leukocytes  formed  rosette-like 
masses  about  the  organisms.  At  first  polymorphonuclear  leukocytes  were  found 
in  such  masses;  later,  the  groups  consisted  entirely  of  macrophages.  Malvoz'^  (1901) 
experimented  with  six  strains  of  yeast,  some  pathogenic  and  some  non-pathogenic. 
The  pathogenic  cultures  he  obtained  from  Sanfelice,  Plimmer,  Curtis,  and  one  he 
isolated  himself  from  an  epithelioma,  calling  it  Blastomyces  E  (BE)  as  a  convenient 
laboratory  name.  His  non-pathogenic  cultures  consisted  of  a  strain  of  Saccharomyces 
ellipsoideus  and  a  powerfully  fermentative  organism  which  he  called  the  yeast  of  Huy 
Rabbits  were  given  two  weekly  injections  of  the  organism  of  Huy,  BE,  and  Sanfelice 
over  a  period  of  months,  and  the  agglutinative  properties  of  the  sera  were  then  tested. 
The  specific  serum  agglutinated  the  organism  of  Huy  in  a  dilution  of  1:50;  organism 
BE,  1:90;  organism  Sanfelice,  1:5.  The  specific  serum  for  the  organism  of  Huy 
agglutinated  S.  ellipsoideus  in  the  same  titre  as  the  organism  of  Huy  itself;    more 


Immunological  Reactions  of  Oidiomycosis  i8i 

weakly  the  organism  of  Plimmer  and  organism  BE;  and  the  organism  of  Curtis  not 
at  all.  The  specific  serum  for  organism  BE  agglutinated  organism  of  Huy  very  slightly. 
The  organisms  of  Huy,  Sanfelice,  and  organism  BE  grew  well  in  normal  and  immune 
serum,  and  grew  on  artificial  media  when  transplanted  after  i  to  24  hours.  The 
author  concluded  that  in  this  case  either  bactericidal  antibodies  were  absent  or  the 
organisms  were  protected  by  their  capsule.  He  suggests  that  the  Bordet-Gengou 
reaction  might  be  of  service  in  deciding  the  presence  or  absence  of  such  antibodies. 

Ricketts"  (1901)  says:  "Professor  Hektoen  has  found  that  the  undiluted  serum 
of  a  dog  which  had  received  successive  inoculations  of  the  organism  from  Case  I 
(cutaneous  oidiomycosis)  causes  gradual  clumping  of  the  organism  diffused  in  broth. 
Several  hours  elapse  before  the  fullest  extent  of  clumping  possible  is  reached.  Organ- 
isms from  other  cases  show  only  a  slight  degree  of  clumping  with  the  same  serum. 
All  organisms  grew  well  in  the  serum  and  the  production  of  mycelium  was  especially 
noticeable.  Normal  dog's  serum  caused  no  clumping  of  any  of  the  organisms;  abun- 
dant mycelium  is  produced  in  all  cases. 

"The  organism  from  the  case  reported  by  Hyde,  Hektoen,  and  Bevan  was  repeat- 
edly inoculated  into  the  abdominal  cavity  of  a  rabbit.  It  was  found  that  the  animal's 
serum  would  cause  fairly  distinct  agglutination  of  the  organism  inoculated." 

Sanfelice^  (1896)  discovered  that  heating  for  30"  at  60°  C.  destroyed  the  viru- 
lence of  his  Saccharomyces  neoformans  for  guinea-pigs,  but  did  not  destroy  its  power 
to  grow  on  artificial  media.  He  endeavored  to  immunize  guinea-pigs  against  this 
organism  (i)  by  the  injection  of  organisms  whose  virulence  had  been  destroyed  by 
heating  to  60°  C.  for  30";  (2)  by  repeated  injections  of  filtered  broth  cultures  of 
various  ages;  and  (3)  by  the  injection  of  the  serum  of  guinea-pigs  and  of  dogs  which 
had  received  repeated  injections  of  either  the  heated  organism  or  the  filtered  culture, 
or  had  recovered  from  the  infection  induced  by  a  dose  of  virulent  organisms.  He 
was  wholly  unsuccessful. 

In  1902  Sanfelice's  was  able  to  immunize  dogs,  cats,  and  rabbits  to  Saccharomyces 
neoformans,  Plimmer's  yeast,  and  to  a  non-pathogenic  yeast  isolated  from  the  air, 
so  that  the  animals  could  withstand,  without  symptoms,  an  intravenous  injection  of 
quantities  of  the  first  two  organisms  which  caused  the  death  of  normal,  control  ani- 
mals. He  had  no  difliculty  in  demonstrating  the  presence  of  a  specific  amboceptor  in 
the  serum  of  such  immune  animals  by  the  fixation  test.  He  was  unable  to  demon- 
strate the  antibody  in  actively  infected  animals  which  subsequently  died  of  the  dis- 
ease. Unfortunately  he  never  took  the  trouble  to  control  his  fixation  experiments  by 
testing  the  effects  of  his  antigen — emulsion  of  yeast — or  of  his  antibodies — inactivated 
immune  and  normal  sera — upon  the  hemolytic  system  which  he  used — serum  of  rab- 
bits immune  to  fowl  corpuscles — a  fact  which  detracts  considerably  from  the  value  of 
his  results.  He  says  that  Malvoz"  demonstrated  antiblastomycetic  amboceptors  by 
the  fixation  reaction  in  1901.  Sanfelice  believed  that  the  immune  sera  caused  a 
change  in  the  yeast  cells  exposed  to  their  action  such  that  the  organisms  assumed  an 
appearance  identical  with  the  so-called  Russell's  fuchsin  bodies  of  malignant  tumors. 

Wlaeff"^  (1902)  immunized  geese  and  donkeys  to  a  yeastlike  organism  which  he 
isolated  from  the  ascitic  fluid  of  a  patient  whose  disease  had  been  diagnosed  as  inop- 
erable abdominal  cancer  with  ascites.  A  piece  of  the  growth,  removed  at  an  explora- 
tory operation,  was  diagnosed  by  Cornil  as  a  typical  cyhndrical-celled  carcinoma. 
The  sera  of  the  immunized  animals  agglutinated  and  dissolved  the  specific  organisms. 
Wlaeff  claimed  that,  following  repeated  injections  of  such  sera,  the  ascitic  fluid  of  this 


1 82  Contributions  to  Medical  Science 

cancer  patient  likewise  developed  the  property  of  agglutinating  and  dissolving  the 
blastomycetes  and  that  the  cancer  improved  as  the  result  of  such  treatment. 

Fabozzi'*  (1905)  concluded  from  his  experiments  that  Saccharomyces  neoformans 
never  causes  the  development  of  tvmiors,  and  is  finally  destroyed  by  phagocytes. 

Christensen  and  Hektoen"  (1906)  say:  "The  character  of  the  lesions  of  blas- 
tomycosis, the  accumulation  of  leukocytes,  the  formation  of  giant  cells,  and  the  pha- 
gocytosis of  blastomycetes — indicates  that  this  is  an  infection  in  which  phagocytosis 
is  an  important  means  of  defense  and  healing.  Certain  preliminary  test-tube  experi- 
ments showed  that  phagocytosis  of  blastomycetes  is  favored  by  the  presence  of  normal 
serum,  and  the  idea  arose  that  it  might  be  possible  to  stimulate  the  greater  formation, 
in  cases  of  blastomycosis,  of  the  body  that  promotes  phagocytosis  (opsonin),  as  well 
as  the  other  antibodies,  by  the  injection  of  blastomycetic  substances  in  a  readily 
absorbable  form.  It  was  thought  that  the  resistant  character  of  the  micro-organisms, 
coupled  with  their  inclosure  in  cellular  exudate  and  granulation  tissue,  possibly  pre- 
vents the  absorption  in  proper  quantities  of  the  substances  necessary  to  call  forth 
strong  immunizing  reactions.  Hence,  in  order  to  hasten,  if  possible,  the  reactions 
that  favor  healing,  we  injected  in  each  of  our  cases  a  sterile  blastomycetic  vaccine 
prepared  by  Dr.  H.  T.  Ricketts  of  the  corresponding  organism.  Unfortunately  the 
patients  left  the  hospital  at  a  time  when  no  conclusions  of  value  could  be  drawn  as  to 
the  results  of  the  vaccine." 

Marco  del  Pont^^  (1907),  using  Endomyces  albicans,  grew  broth  cultures  with 
increasing  doses,  first,  of  normal,  and  later,  of  immunized  rabbit's  serum.  The  devel- 
opment of  a  mycelium  indicated  that  the  medium  was  unfavorable.  After  the  60th 
transfer  the  yeast  grew  normally  in  undiluted  immune  serum.  It  retained  this  faculty. 
The  immunity  was  specific;  yeast  habituated  to  rabbit  serum  did  not  grow  in  the  serum 
of  the  dog,  goat,  or  rat.  The  immunized  fungus  had  lost  its  virulence.  The  author 
thought  that  the  immunity  to  the  serum  might  be  due  to  the  development  of  a  muci- 
laginous capsule  by  the  organism;  he  also  thought  that  a  substance  anti-sensibilisatrice 
might  be  produced  by  the  micro-organism. 

Widal,  Abrami,  Joltrain,  Brissaud,  and  WeilP'  (1910)  have  found  that  the  blood 
serum  from  cases  of  sporotrichosis  possesses  the  power  of  agglutinating  the  spores — 
but  not  the  cell  bodies — of  the  Sporotrichum  schenckii  in  dilutions  of  from  i :  300 
to  1 :  800.  The  agglutinability  of  the  spores  varies  with  the  age  of  the  culture  and 
with  the  kind  of  medium,  but  not  with  the  strain  of  the  organism  (10  strains  were  com- 
pared). The  Bordet-Gengou  reaction  was  constant.  Among  168  normal  subjects 
and  subjects  suffering  from  other  diseases,  they  found  an  occasional  agglutination  or 
fixation  reaction,  but  both  never  occurred  in  the  same  individual.  The  two  reactions, 
therefore,  control  each  other.  Experimentally,  two  dogs  and  four  rabbits  were  used. 
In  dogs  it  was  easy  to  produce  infection  with  the  organisms,  and  the  agglutination  and 
fixation  reactions  were  constant  and  marked.  In  rabbits,  intravenous  injections  of 
large  quantities  failed  to  cause  infection.  The  agglutinative  power  of  the  serum 
increased  appreciably;  it  varied  from  1:10  to  1:30  in  the  different  rabbits  before 
injection,  and  rose,  following  injection,  as  high  as  i :  3,000  in  one  case  and  to  but  i  :300 
in  another.  The  reaction  of  fixation,  which  was  tested  parallel  with  the  agglutina- 
tion tests,  gave  "very  incongruous  results."  No  further  details  of  these  results  are 
stated. 

They  found  that  the  agglutination  and  fixation  reactions  disappeared  in  man 
after  the  infection  was  conquered.     The  fixation  reaction  disappeared  more  rapidly 


Immunological  Reactions  of  Oidiomycosis  183 

than  the  other.  They  concluded  that,  so  long  as  both  are  found,  an  active  lesion  is 
indicated;  the  disease  is  not  really  cured. 

Co-reactions. — The  organisms  tested  which  did  not  give  co-agglutination  and 
co-fixation  reactions  with  immune  sporothrix  serum  and  vice  versa  were  the  tricho- 
phytons, the  organisms  of  Erythrasma,  Favus,  and  of  animal  aspergillosis.  The  re- 
actions of  co-fixation  and  co-agglutination  were  obtained  with  organisms  isolated  from 
cases  of  actinomycosis  and  thrush  (Oidium  albicans).  The  serum  from  the  case  of 
infection  with  Oidium  albicans  agglutinated  the  spores  of  the  sporothrix  3  times  as 
strongly  as  it  did  the  oidium  itself,  which  was  agglutinated  only  in  dilutions  of  1:10 
to  1:50.  The  co-fixation  reaction  was  positive  with  the  following  cultures  of  yeast: 
the  organism  of  Curtis,  of  Blanchard,  of  Plimmer,  O.  luteum,  S.  granulatus,  S.  litho- 
genes,  and  S.  caprae,  using  sera  from  cases  of  sporotrichosis,  actinomycosis,  and  thrush. 
The  organism  of  actinomycosis  itself  was  not  agglutinated  by  the  specific  serum. 

Rothe^*  (1909),  noting  the  preliminary  reports  of  Widal,^'  tested  the  agglutinating 
power  of  the  blood  serum  from  two  cases  of  actinomycosis  upon  the  spores  of  the  sporo- 
thrix and  found  them  positive  in  dilutions  of  i :  160  and  i :  200  respectively. 

MATERIALS   AND    TECHNIC, 

The  organism  used  was  obtained  in  1905  by  Dr.  Rosenow  from 
a  case  of  oidiomycosis  in  the  Presbyterian  Hospital  of  Chicago. 
It  had,  therefore,  been  growing  on  artificial  media  for  three  years 
before  the  present  work  was  begun.  The  stock  culture  was  grown 
on  a  I  per  cent  glucose,  i  per  cent  acid  agar,  and  also  in  nutrient 
broth  of  similar  sugar  and  acid  content.  On  acid-glucose-agar, 
at  room  temperature,  the  organism  grows  in  the  form  of  long 
hyphae  which  burrow  peripheralward  through  the  superficial 
layers  of  the  agar.  No  aerial  hyphae  are  developed  excepting  in 
those  cultures  which  have  been  started  in  the  incubator  and  are 
later  placed  at  room  temperature.  The  characteristics  of  these 
hyphae  will  be  discussed  later. 

In  acid-glucose-broth,  at  room  temperature,  the  organism  grows 
in  the  form  of  fluffy  balls,  the  size  which  the  latter  may  reach 
apparently  being  limited  only  by  the  volume  of  the  broth,  the 
capacity  of  the  retaining  vessels,  and  the  number  of  colonies  which 
develop.  There  seems  to  be  no  production  of  gas.  In  old  cul- 
tures a  heavy  membrane  forms  on  the  surface  of  the  broth,  and 
the  medium,  though  remaining  clear,  assumes  a  dark  amber  color. 
Under  the  low  power  of  the  microscope  in  a  hanging-drop  prepara- 
tion kept  at  room  temperature  and  first  observed  October  20,  1908, 
one  of  these  "fluffy  balls"  appeared  as  a  mass  of  mycelial  threads 
arranged  in  two  zones,  a  central  mass  of  tangled  threads  and  granu- 


184  Contributions  to  Medical  Science 

lar  material  too  thick  for  light  to  pierce  and  so  not  discernible  in 
detail,  and  a  peripheral  zone  of  radiating  threads.  These  threads 
presented  a  rather  homogenous  appearance,  were  quite  refractive 
to  light,  and  possessed  a  slightly  greenish  color  by  transmitted 
light.  Their  central  ends  were  lost  in  the  central  mass;  their 
peripheral  ends  were  bluntly  rounded.  Branches  were  few  and 
these  few  were  found  toward  the  base  of  the  radiating  threads; 
the  branches  were  equal  in  diameter  to  the  parent  stem,  were  of 
uniform  thickness  throughout  their  length,  for  the  most  part,  and 
were  given  off,  sometimes  at  right  angles,  but  usually  at  an  angle 
of  about  45°.  The  contour  of  the  angle  was  not  that  of  a  sharp 
corner,  but  rounded.  No  segmentation  of  the  h>"phae  nor  lateral 
conidia  were  observed.  No  distinct  cell  wall  appeared.  On 
October  22,  two  days  later,  the  following  notes  were  made:  Hyphae 
have  at  least  doubled  in  length;  three  zones  can  now  be  distin- 
guished in  the  colony:  (i)  a  central,  practically  opaque  zone; 
(2)  a  middle  zone  of  branched,  interlacing  hyphae  bearing  lateral 
conidia;  most  of  these  hyphae  show  marked  but  somewhat  irregu- 
lar segmentation;  and  (3)  a  peripheral  zone  similar  to  the  one 
described  on  October  20.  The  lateral  conidia  arise  by  short  stalks 
from  alternate  sides  of  the  hyphae,  one  conidium  to  each  segment. 
In  some  cases  no  structures  are  discernible  within  the  conidia; 
at  other  times  they  seem  to  be  filled  with  sharply  defined  spherules. 
The  segments  consist  of  oblong,  clear  areas  separated  from  each 
other  by  discs,  cubes,  and  cylinders  of  a  homogenous,  highly  refract- 
ive, greenish  substance  very  similar  in  appearance  to  the  material 
in  the  young  hyphae  described  two  days  before.  The  peripheral 
ends  of  the  hyphae  and  those  intermediate  portions  in  which  the 
segmentation  is  but  slightly  marked  contain  scattered  small, 
spherical,  highly  refractive  bodies  which  seem  to  be  attached  to 
the  inner  side  of  the  double-contoured  cell  wall  which  has  now 
become  visible.  The  hyphae  are  reaching  far  out  into  the  vase- 
line by  which  the  cover  glass  is  attached. 

November  29. — No  change  in  method  of  development. 

In  agar  hanging-drop  preparations  kept  at  room  temperature  es- 
sentially the  same  appearances  are  obtained  as  in  the  broth.  Some- 
times the  lateral  conidia  appear  to  be  filled  with  sharply  out- 


Immunological  Reactions  of  Oidiomycosis  185 

lined,  homogenous  spherules.  If  such  conidia  are  kept  under 
observation  for  a  few  weeks,  the  spherules  can  be  seen  to  gradually 
fuse  into  larger  and  larger  masses  until  finally  the  conidial  content 
consists  of  a  single  homogenous  mass.  A  somewhat  similar  con- 
dition has  been  described  by  Bowen  and  Wolbach.^"  In  the  case 
described  by  these  authors,  bodies  were  found  on  agar  tubes, 
which  were  filled  with  refractive  spherules.  When  these  bodies 
were  placed  on  fresh  media  the  spherules  fused  before  mycelial 
formation  began. 

On  acid-glucose-agar  at  37°  C,  the  organism  grew  exclusively 
in  the  budding  form,  that  is,  like  yeast,  for  about  fourteen  months, 
when  it  rather  suddenly — in  the  course  of  a  month  or  six  weeks — 
developed  a  propensity  for  growing  in  the  hyphal  form  to  which 
it  has  clung  tenaciously  since.  While  growing  in  the  budding  form 
the  colonies  which  formed  on  the  agar  were  soft,  yellowish-white, 
raised,  and  button-shaped,  consisting  of  a  flat  central  area  sur- 
rounded by  a  thickened,  rounded  border.  They  were  circular  in 
outhne  and  from  1-7  mm.  in  diameter.  The  individual  cells  were 
from  10-30  At  in  diameter. 

With  the  assumption  of  the  hyphal  form  of  growth,  the  organ- 
isms became  prone  to  the  development  of  aerial  hyphae.  This 
tendency  was  especially  noticeable  in  cultures  kept  at  about 
34.5°  C. ;  36-37°  C.  and  room  temperature  (about  19-20° C.)  appeared 
to  be  very  much  less  favorable  to  their  production,  excepting  in 
cultures,  as  mentioned  above,  which  had  been  transferred  from  the 
incubator  to  room  temperature.  To  the  naked  eye,  the  hyphae — 
developed  at  34.5°  C. — appear  as  very  delicate  stalks  perhaps  a 
milHmeter  in  height  (estimated),  white  by  transmitted  light, 
and  having  a  tendency  to  assume  a  silvery  sheen  by  reflected  light. 
In  the  mass,  they  present  a  dead  white,  somewhat  fuzzy  appear- 
ance, resembling  a  piece  of  high  grade,  heavy  filter-paper.  About 
the  edges  of  the  growth,  where  the  hyphae  are  less  numerous,  the 
cultures  have  a  ''woolly"  appearance.  In  this  condition  the  culture 
is  less  adherent  to  the  medium  than  at  earlier  stages — the  hyphae 
begin  to  appear  about  10  days  to  two  weeks  after  inoculation — 
and  may  be  peeled  off  in  a  single  layer  taking  with  it  pieces  of  agar. 
At  a  few  scattered  points  hyphae  may  be  seen  which  are  three  or 


1 86  Contributions  to  Medical  Science 

four  times  the  ordinary  length  and  project  above  the  general  level 
at  odd  angles,  like  the  poles  from  the  top  of  an  Indian  wigwam — 
probably  the  "porcupine"  appearance  mentioned  by  Hamburger." 

Under  the  microscope  the  h}phae  appear  as  slender,  hollow  rods, 
I  to  2  M  in  diameter,  with  a  very  delicate  wall.  They  contain  a 
very  fine  granular  substance  in  addition  to  quite  highly  refractive, 
homogenous,  greenish  spherules  which  vary  in  diameter  from  a  frac- 
tion of  a  micron  to  one  micron.  Sometimes  the  hyphae  are  seg- 
mented and  bear  lateral  and  terminal  conidia,  as  well  as  occasional 
enlargements  of  the  individual  segments,  in  the  course  of  the  tube 
itself.  The  conidia  and  other  swelHngs  are  from  5  to  lo  /"•  in  diame- 
ter and  contain,  usually,  one,  and  occasionally  3  or  4  spherules,  simi- 
lar in  appearance  to  those  noted  in  the  bodies  of  the  hyphae,  but 
of  considerably  larger  size,  4  to  7  /^  in  diameter.  In  the  larger 
conidia,  the  double-contoured  membrane  which  forms  their  wall 
is  very  conspicuous.  Some  of  the  largest  conidia  are  empty; 
their  walls  are  nearly  i  At  in  thickness  and  present  breaks  in  their 
continuity.  Free  in  the  suspending  fluid,  0.85  per  cent  NaCl 
solution,  about  the  hyphae  under  examination,  are  numbers  of 
spherules  exactly  similar  in  appearance  to  those  described  within 
conidia.  Their  origin  from  the  latter  seems  probable.  They 
frequently  present  a  marked  Brownian  movement. 

In  acid-glucose-broth  cultures  a  granular  sediment  forms  in 
the  course  of  a  week  or  ten  days,  the  overlying  fluid  remaining 
clear.  On  shaking,  the  tube  becomes  diffusely  clouded  but  resumes 
its  original  appearance  after  standing  a  short  time.  Microscopi- 
cally the  typical  budding  yeast  forms  appear.  Occasionally 
elongated  forms  suggesting  h>phae  are  found.  The  same  change 
has  overtaken  the  broth  cultures  that  has  been  mentioned  as  occur- 
ring in  the  agar  cultures,  that  is,  the  organisms  no  longer  grow 
in  the  budding  form  but  have  reverted  to  the  growth  character- 
istic of  room  temperature,  namely,  the  mycelial  form.  Whether 
or  not  it  will  be  possible  to  induce  the  organism  to  resume  the 
former  mode  of  growth  at  incubator  temperature  (37°  C.)  is  a 
question  which  must  be  left  for  further  observation. 

No  attempt  has  been  made  to  determine  the  finer  details  of 
the  biology  and  morphology  of  this  organism,  since  it  is  believed 


Immunological  Reactions  of  Oidiomycosis  187 

that  enough  has  been  said  to  warrant  the  assumption  that  we  are 
deahng  with  an  organism  which  undoubtedly  belongs  to  that  genus 
of  the  pathogenic  mould-fungi  placed  by  Ricketts,"  provisionally, 
among  the  oidia,  and  which  therefore  may  be  considered  a  fair 
sample  of  the  parasites  responsible  for  oidiomycosis  (blastomycosis) 
in  man. 

Besides  the  organism  itself,  a  so-called  oidiomycetic  "extract" 
was  made  use  of  in  the  following  experiments.  This  "extract" 
was  made  according  to  a  method  which  has  been  in  use  in  this 
laboratory  for  a  number  of  years,  and  which  is  as  follows: 

The  organism,  in  order  to  obtain  considerable  quantities,  is  planted  on  a  large 
covered  plate  or  in  flat-sided,  wide-mouthed  bottles.  When  a  good  growth  has 
appeared,  which  may  take  from  three  weeks  to  a  month,  it  is  scraped  off  by  means 
of  a  glass  or  platinum  hoe  and  placed  in  a  desiccator  where  it  is  allowed  to  dry.  The 
dried  material  is  weighed,  then  placed  in  a  porcelain-ball  mill  together  with  an  equal 
volume  of  sterile  sand  and  about  lo  c.c.  of  sterile  0.85  per  cent  salt  solution  and  ground 
for  a  couple  of  hours.  Salt  solution  in  small  quantities,  5  c.c,  is  then  added  at  short 
intervals  until  the  volume  of  fluid  is  equal  to  about  50  c.c.  The  liquid  is  drawn  off, 
centrifugated,  the  supernated  fluid  poured  into  a  sterile  bottle  or  other  suitable  recep- 
tacle, and  the  solid  portion,  which  still  contains  large  numbers  of  yeast  cells,  is  returned 
to  the  mill  and  reground.  This  process  is  repeated  until  microscopical  examination 
fails  to  reveal  unbroken  cells  in  the  centrifugated  sediment.  The  various  fluid  portions 
are  added  together  and  the  total  volume  made  up  to  such  a  point  that  every  100  c.c. 
represents  one  gram  of  the  dried  organism.  As  a  preservative,  0.5  per  cent  carbolic 
acid  or  0.3  per  cent  chloroform  is  then  added.  As  a  further  precaution  the  "extract" 
is  kept  in  the  ice-box.  If  one  wishes  a  fresh  extract  the  moist  organisms  may  be  used. 
It  has  been  shown  by  experiment  that  the  yeast  loses  about  }  ^  of  its  weight  when 
dried.  Therefore  if  one  grinds  1 2  grams  of  fresh,  moist  organisms  and  wishes  to  make 
a  I  per  cent  solution  comparable  to  the  preserved  extracts,  he  makes  the  total  volume 
of  extract  100  c.c.  This,  of  course,  is  not  a  method  of  great  accuracy,  owing  to  the 
variations  in  the  amount  of  water  which  the  moist  organisms  contain,  but  it  seems 
fairly  satisfactory. 

Upon  standing  any  length  of  time  a  white,  amorphous  precipitate  separates  out 
of  the  extract  and  settles  to  the  bottom.  It  does  not  go  into  solution  again  when 
mixed  with  the  overlying  fluid.  The  extract  itself  has  a  yellowish,  opalescent  appear- 
ance, which  tends  to  clear  somewhat  on  shaking  with  ether.  It  possesses  a  markedly 
yeasty  smell.  The  following  rough  qualitative  analysis  was  made  on  "Ext.  BIR 
1/19/09."     The  results  are  typical  of  those  observed  in  all  cases  examined. 

To  10  c.c.  of  the  extract  were  added  30  c.c.  of  absolute  alcohol,  and  the  mixture 
was  allowed  to  stand  in  the  ice-box  for  30  minutes.  At  the  expiration  of  that  time,  a 
fairly  heavy,  whitish,  flocculent  precipitate  had  formed,  which  showed  a  tendency  to 
remain  suspended  in  the  liquid  in  large  and  small  loose  clumps.  The  material  was 
filtered.  The  filtrate  was  evaporated  to  dryness  at  a  temperature  not  over  65°  C. 
and  yielded  a  yellow,  gummy-looking  material  which  possessed  a  marked  sweetish, 
yeasty  smell.    This  material  was  allowed  to  stand  over  night  at  room  temperature. 


1 88  Contributions  to  Medical  Science 

The  precipitate  on  the  filter  paper  was  washed  with  30  c.c.  of  distilled  water  in  which 
it  seemed  to  be  readily  taken  up,  giving  the  water  a  whitish,  cloudy  appearance.  It 
had  no  characteristic  odor.  Under  the  microscope,  the  aqueous  solution  showed  a 
fine,  granular  debris,  but  no  crystalline  suspension;  the  residue  from  the  evaporated 
alcoholic  extract  was  amorphous  and  of  a  yellow  tinge. 

The  aqueous  solution  was  tested  for  dextrin,  starch,  and  reducing  sugars  with 
negative  results.    The  protein  color  and  coagulation  tests  resulted  as  follows: 

Heller's  nitric  acid  test  negative 

Boiling  with  glacial  acetic  " 

Millon's  reagent  " 

Xanthroproteic  test  " 

Adamkiewicz  +very  sharp 

Biuret  ?  very  slight  if  any 

Liebermann  negative 

Hopkins-Cole  +very  sharp 

H2SO4  (conc.)+sugar  negative 

Similar  tests  applied  to  the  whole  extract  gave  similar  results. 

The  yellow  residue  from  the  alcoholic  extract  was  soluble  in  ether  and  chloroform 
and  was  not  precipitated  from  the  chloroform  solution  by  acetone.  It  gave  a  negative 
Salkowski's  test  for  cholesterol.     Further  analysis  was  not  attempted. 

No  experiments  were  undertaken  with  killed  organisms,  the 
living  organisms  and  the  extract  alone  being  used  in  the  immuno- 
logical work. 

As  a  matter  of  routine,  injections  of  living  organisms  were  made 
into  the  peritoneal  ca\dty;  the  pleural  cavity  was  used  a  few  times. 
Intravenous  injections  are  inconvenient  in  the  guinea-pig,  while 
subcutaneous  injections  are  open  to  the  objection  that  the  result- 
ing subcutaneous  nodules  early  cause  necrosis  and  sloughing  of 
the  overlying  skin,  thus  not  only  giving  splendid  opportunities  for 
secondary  infections  but  tending  to  scatter  virulent  mould-fungi 
about  the  cages,  which  was  regarded  as  dangerous. 

The  extract  was  injected  subcutaneously  or  intraperitoneally 
according  to  the  seeming  needs  of  the  particular  experiment  in 
which  it  was  being  used. 

oidiomycosis  (blastomycosis)  in  the  gulnea-pig. 

Dosage. — In  a  series  of  experiments  carried  out  about  three 
years  previously  with  this  same  organism.  Dr.  Ricketts  had  deter- 
mined roughly  its  virulence  for  guinea-pigs.  He  found  that  0.3 
of  a  gram  of  an  agar  plate  culture,  grown  at  incubator  tempera- 
ture, injected  into  the  peritoneal  cavity  of  a  375-400  gm.  guinea-pig 


Immunological  Reactions  of  Oidiomycosis  189 

would  cause  death  in  about  35  days,  with  generahzed  oidiomycosis, 
especially  in  the  abdominal  and  thoracic  viscera. 

In  view  of  the  prolonged  course  of  the  disease,  even  following 
the  injection  of  such  large  doses  as  that  mentioned,  it  did  not 
seem  so  essential  for  present  purposes  to  determine  a  uniformly 
fatal  dose  as  to  find  out  what  dosage  would  give  a  constant  clini- 
cal picture,  and,  at  the  same  time,  would  not  require  a  month  or 
six  weeks  for  development.  With  this  in  view  a  series  of  42  guinea- 
pigs  were  injected  with  quantities  of  moist  organisms  from  agar 
culture,  the  dosage  varying  from  0.5  to  o.oooi  of  a  gram.  Each 
pig  was  examined  daily  with  reference  to  weight,  temperature, 
and  to  changes  which  might  be  revealed  by  inspection  or  palpation. 
The  following  general  conclusion  was  drawn:  There  is  no  con- 
stant symptom  by  means  of  which  one  may  diagnose  oidiomycosis 
in  the  guinea-pig  during  life.  The  weight  is  an  unreliable  guide, 
it  varies  with  the  abundance  of  food  and  the  length  of  time 
which  elapses  between  feeding  and  the  taking  of  weights.  Again, 
even  if  the  animal  develops  an  apparently  fatal  infection,  the 
weight  before  death  may  approximately  equal  that  before  inocu- 
lation, or,  if  it  does  decrease,  the  major  portion  of  the  loss  occurs 
during  the  last  week  or  so  of  life.  The  temperature  is  also  unsat- 
isfactory. In  the  majority  of  cases  when  the  oidiomycosis  cannot 
be  doubted,  the  temperature  runs  an  absolutely  normal  course. 
When  it  does  follow  an  abnormally  high  curve,  it  is  difficult  to 
exclude  secondary  infection,  or  the  possible  efi"ect  of  occlusion  of 
excretory  ducts  (that  is,  ureter,  seminal  vessels,  urethra). 

If  one  compares  the  effects  in  males  and  females,  however,  he 
finds  that  there  is,  symptomatically  at  least,  one  marked  differ- 
ence. In  males  with  doses  as  low  as  o.ooi  gm.  one  usually  can 
detect  small  nodules  (o .  5-2  mm.  in  diameter)  in  the  testicles  within 
seven  to  ten  days  after  inoculation;  nodules  can  practically 
always  be  found  with  doses  of  o.oi  gm.  within  five  to  seven  days. 
With  doses  of  o.i  gm.  or  over,  nodules  not  only  occur  regularly 
but  they  are  of  good  size  and  the  infection  may  have  a  fatal  out- 
come. In  the  female  pig  there  is  absolutely  no  sure  way  of  diag- 
nosing oidiomycosis  during  life  except  to  open  the  abdominal 
cavity  and  inspect  the  contents.     One  might  think  of  deep  pal- 


iQo  Contributions  to  Medical  Science 

pation,  but  how  distinguish  nodules  from  feces  ?  We  have  found 
it  practically  impossible  in  most  cases.  Of  great  assistance  in 
determining  the  severity  of  the  infection — and  this  may  be  applied 
to  both  sexes  ahke — is  the  "look"  and  "feel"  of  the  animals.  By 
the  term  "feel"  well  is  meant  that  when  the  animal  is  handled 
the  muscles  are  found  to  have  their  normal  firmness  and  tone. 
In  fresh  guinea-pigs  which  have  not  been  handled,  the  tense,  hard, 
wiry  feeling  of  the  muscles  is  very  noticeable.  After  the  animals 
have  been  manipulated  daily  for  a  week  or  so,  they  evidently 
become  used  to  it  and  relax  readily  when  they  are  picked  up.  In 
this  state  the  muscles  are  soft  and  pliable  but  have  not  lost  their 
tone.  If  such  pigs  become  the  victims  of  a  chronic  wasting  dis- 
ease, this  normal  muscular  tone  is  lost;  the  muscles  become 
decidedly  flabby  to  the  touch  so  that  the  animal  feels  like  nothing 
so  much  as  the  time-honored  "dish-rag."  This  "feel"  may  be 
present  before  the  animal's  weight  has  fallen  off  appreciably. 
The  animal  usually  appears  thin  and  we  say  it  looks  sick,  but  this 
appearance  is  due  more  to  the  staring  coat  and  the  "hunched-up" 
attitude  which  the  animal  assumes  than  to  a  real  emaciation. 
These  symptoms,  of  course,  are  characteristic  of  cachectic  condi- 
tions in  general  and  are  not  specific.  It  should  be  mentioned  that 
the  development  of  nodules  at  the  point  of  inoculation  noted  in 
some  of  the  pigs  was  regarded  as  the  result  of  faulty  technic  and 
could  be  avoided  to  a  considerable  extent  by  rinsing  the  needle 
in  water  before  making  the  injection. 

For  the  remainder  of  this  work,  in  view  of  the  above  facts,  the 
taking  of  temperatures,  the  recording  of  weights,  and  the  use  of 
female  guinea-pigs  were  eliminated  excepting  in  special  cases. 
Male  pigs  were  used  as  a  matter  of  routine  and  their  condition  was 
adjudged  by  the  results  of  careful  palpation  of  the  testicles  and 
by  their  general  "look"  and  "feel."  o.i  gm.  of  moist  agar  cul- 
ture was  adopted  as  the  standard,  surely  infective  dose. 

The  difference  in  the  results  of  intraperitoneal  inoculation  in 
male  and  female  guinea-pigs  is  very  marked  from  the  symptomato- 
logical  standpoint.  Take  guinea-pig  75  and  guinea-pig  81,  for 
example.  Each  received  the  same  dose  from  the  same  culture 
on  the  same  day.     Guinea-pig  75  (male)  became  weak  and  flabby 


Immunological  Reactions  of  Oidiomycosis  191 

to  the  touch  ^vith  a  staring  coat  and  enormously  swollen  scrotum 
which  finally  ulcerated.  Guinea-pig  81  grew  fat  and  sleek  and 
it  was  only  at  autopsy  that  signs  of  the  infection  could  be  demon- 
strated. Some  of  the  possible  reasons  for  this  diflference  will  be 
discussed  later  as  will  also  the  question  of  localization  of  infection, 
and,  in  a  general  way,  the  course  of  the  disease  and  possible  treat- 
ment. 

active  immunization. 

In  the  endeavor  to  establish  an  active  immunity  in  guinea-pigs, 
use  was  made  of  the  living  organism  and  of  the  extract.  In  the 
case  of  the  living  organism,  the  method  pursued  for  the  greater 
part  of  the  time  was  to  reinoculate  a  pig  as  soon  as  he  had  made 
an  apparently  complete  recovery  from  his  previous  infection,  and 
then  to  study  the  development  of  the  new  infection  as  regards 
(i)  the  time  of  the  appearance  of  nodules  in  the  testicles,  their 
size,  consistency,  and  the  rate  at  which  they  disappeared,  (2)  the 
development  of  palpable  nodules  in  the  abdominal  cavity,  and 
(3)  the  "look"  and  "feel"  of  the  animal.  The  results  of  such 
observations  had  led  to  the  belief  that  it  made  practically  no  differ- 
ence how  many  times  an  animal  was  subjected  to  oidiomycetic 
infection  and  recovered,  he  always  retained  his  original  suscepti- 
bility. In  order  to  put  this  conclusion  to  a  thorough  test  the 
following  experiment  was  performed.  All  the  living  pigs  which 
had  recovered  from  previous  injections  were  gathered  together 
with  ten  normal  pigs  of  about  the  same  average  size  and  all  were 
given  an  equal  dose  of  living  oidiomycetes  (approximately  o.i 
gm.)  and  the  results  carefully  watched.  The  previous  history 
of  the  so-called  "immune"  animals  is  given  in  Table  i. 

On  comparing  the  results  of  the  inoculation  in  the  immune 
and  the  control  guinea-pigs,  some  sHght,  but  quite  well  marked 
differences  were  noted.  First,  regarding  the  course  of  the  infec- 
tion, it  is  a  fact  worthy  of  remark,  that  in  so  far  as  the  male  pigs 
of  the  two  series  are  concerned,  the  immune  animals  made  a  decid- 
edly more  rapid  recovery  than  the  controls,  as  judged  by  the  rate 
of  disappearance  of  the  nodule  from  the  testicles.  The  female 
pigs  offer  no  accessible  basis  for  comparison.     If  we  examine  the 


192 


Contributions  to  Medical  Science 


temperature  charts  of  the  immune  and  normal  animals  before  and 
after  injection  we  again  find  differences.  The  average  tempera- 
ture for  the  immunized  animals  for  the  three  days  prior  to  the 
inoculation  on  which  temperatures  were  observed,  that  is,  the 
19th,  20th  and  23d,  is  103.29°+  F.;  that  for  the  control  animals 
is  103.31°+  F.  The  average  of  the  observed  temperatures  of  the 
immunized  animals  on  the  days  immediately  following  the  injec- 
tion (the  24th,  25th,  and  27th)  is  104.27°+  F.;  that  of  the  control 


History  of  ' 

TABLE 
'Immune' 

I. 

'  Guinea-Pigs. 

GUINEA- 

PlG  No. 

No.  Times 
Previous- 
ly  Inocu- 
lated 

Average 
Interval 

BETWEEN 

Inocula- 
tions 

Time 

Elapsed 

Since  Last 

Dosage  (in  gm.) 

Inocu- 
lated 

I  St  Dose 

2d  Dose 

3d  Dose     1    4th  Dose 

3 
4 

I 

4 

4 
3 
4 
2 
2 
2 
2 
2 
3 

6 1  days 

24    " 

24    " 
24    " 
33     " 

24  " 

24 

25  " 

2S      " 
25      " 
32      " 

68  davs 

0.1 
0.1 

0.1 
0.1 
0.1 
0.1 

0.1 

72 

68    ' 
166    ' 
68    ' 
68    ' 
68    ' 
68    ' 
68    ' 
240    ' 
50    ' 
50    ' 
50 
68    ' 

, 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

cx>5 
S 

OOI 

0001 
S 

0 

0 
0 
0 
0 
0 
0 
0 
0 
0 

I 

75 

80 

0  1 

81 

83 

O.l 

47 

93 

94 

95 

69 

guinea-pigs  for  the  same  period  is  103.49°+  F.,  or  practically 
104.3°  F.  in  the  one  case  and  103.5°  '^^  the  other — a  difference  of 
nearly  1°  F.  To  be  sure,  this  difference  is  small,  but  in  view  of  the 
fact  that  the  temperatures  of  the  two  groups  of  pigs  were  almost 
identical  before  inoculation,  while  following  the  inoculation  the 
rise  was  confined  almost  entirely  to  one  group,  it  would  seem 
that  one  would  be  justified  in  considering  such  a  rise  a  posi- 
tive reaction  of  the  animal  organism  against  the  injected  oidio- 
mycetes.  Apparently,  then,  a  low  grade  of  immunity  is  developed 
in  guinea-pigs  by  the  intraperitoneal  inoculation  of  living  organ- 
isms. As  symptomatological  evidence  of  the  immunity — which 
seems  to  be  emphatically  a  relative  immunity — we  have  the  more 
rapid  disappearance  of  the  lesions  following  infective  doses  and 
a  small  but  fairly  decided  rise  in  temperature  on  the  days  imme- 
diately following  the  inoculation.  These  reactions  may  be  mani- 
fest as  long  as  240  days  after  the  last  inoculation  (guinea-pig  49) 


Immunological  Reactions  of  Oidiomycosis 


193 


or,  allowing  the  animal  40  days  in  which  to  recover  from  the 
immunizing  inoculation,  a  liberal  margin,  the  acquired  power  to 
react  against  renewed  infection  in  a  guinea-pig  which  has  recovered 
from  two  moderate  injections  may  persist  at  least  200  days. 

A  large  series  of  guinea-pigs  were  given  repeated  injections  of 
the  extract.  Of  this  series  but  four  pigs  were  tested  by  the  injec- 
tion of  li\dng  organisms.     Their  histories  follow: 

TABLE  2. 


No.  of  Guinea-Pig 


No.  of  Injec- 
tions 


Interval  be- 
tween First 
and  Last  In- 
jection 


Interval  be- 
tween Last  In- 
jection and 
Day  Tested 


Average  Vol- 
ume of  Injec- 
tion 


Route 


13 
37 
38 
S6 


178  days 
10s    " 
los    " 
91    " 


63  days 

77  " 
77  " 
63     " 


About  I .  s  c.c. 
2  c.c. 


Intraperitoneal 


Each  of  these  four  pigs,  together  with  each  of  seven  control 
animals,  was  given  o.i  gm.  BIR,  intraperitoneally.  All  of  the 
control  animals  developed  severe  infections,  large  nodules  in  the 
testicles,  weakness,  staring  coat,  etc.;  and  one  of  them  died,  post- 
mortem examination  revealing  generaUzed  oidiomycosis.  Of  the 
immune  animals,  but  one  developed  testicular  lesions — a  small, 
solitary  nodule;  all  presented  rather  large,  fairly  hard,  intra- 
abdominal masses,  but  no  other  symptoms.  The  subsequent 
history  of  the  immune  pigs  follows.  Guinea-pig  13  was  bled  to 
death  on  December  3,  1909,  102  days  after  injection.  At  autopsy 
the  seminal  vessels  and  portions  of  the  small  intestines  were  found 
adherent  to  a  fibrous  nodule  on  the  anterior  abdominal  wall, 
within  which  was  fluid  detritus  containing  oidiomycetic  forms; 
cultures  were  negative.  Microscopic  sections  show  oidiomycetes. 
No  other  abnormalities. 

Guinea-pig  37  died  January  19,  1910,  149  days  after  injection. 
At  autopsy  a  mass  of  dense  fibrous  adhesions  was  found  about  the 
point  of  inoculation.  In  the  center  of  this  mass  was  a  cavity  i  cm. 
in  length  and  0.5  cm.  in  width  and  about  i  to  2  mm.  deep  in  which 
was  fluid  detritus.  Testicles  were  normal.  No  oidiomycetes 
were  found,  microscopically,  in  the  contents  of  the  nodule.  The 
cause  of  death  was  pneumonia  and  pericarditis.  No  oidiomycetes 
were  recovered  in  cultures. 


194  Contributions  to  Medical  Science 

Guinea-pig  38  was  found  dead  on  March  14,  1910,  203  days  after 
injection.  Autopsy  showed  pneumonia  and  old  fibrous  adhesions 
about  the  point  of  injection.     Testicles  were  normal. 

Guinea-pig  56  was  killed  March  14,  1910,  203  days  after  injec- 
tion. Autopsy  disclosed  an  oval  fibrous  sac  about  i  cm.  in  greatest 
length  attached  to  the  anterior  abdominal  wall.  Adherent  to  the 
sac  were  the  seminal  vesicles  and  portions  of  the  small  intestines. 
Within  the  sac  was  fluid  detritus;  microscopically  forms  very 
similar  to  oidiomycetes  were  observed;  unfortunately  these 
bodies  were  soluble  in  ether.  Cultures  were  negative  as  regards 
oidiomycetes. 

One  cannot  argue  from  these  experiments  that  the  prolonged 
immunization  with  the  ''extract"  resulted  in  the  development 
of  such  a  high  grade  of  immunity  as  would  lead  to  the  absolute 
walling  off  of  infective  doses  of  organisms  at  or  near  the  point  of 
inoculation,  for  it  is  not  at  all  certain  that  those  inoculations  may 
not  have  been  made  into  previously  existing  adhesions,  resulting 
from  the  numerous  immunizing  injections,  which  could  not  be 
detected  by  external  examination.  On  the  other  hand,  in  view 
of  the  fact  that  most  of  the  control  animals  developed  marked 
infection,  one  of  them  dying,  and  that  the  nodules  which  appeared 
in  the  testicles  of  one  of  the  immune  animals  vanished  with  con- 
siderable celerity,  it  seems  justifiable  to  assume  that  a  certain 
degree  of  immunity,  at  least,  was  manifested.  Another  point  of 
interest  which  will  be  considered  later  was  the  finding  of  mor- 
phologically typical  oidiomycetes  in  stained  sections  from  guinea- 
pig  13.  Apparently  this  pig's  immunity,  whatever  it  may  have 
been,  was  not  of  a  markedly  lytic  t3^e. 

EXPERIMENT  TO  TEST  THE  EFFECT  OF  THE  INJECTION  OF  " EXTRACT" 
INTO   INFECTED   ANIMALS. 

Eggers  has  shown  {vide  infra)  that  repeated  intraperitoneal 
injections  of  oidiomycetic  extract  seem  to  decrease  the  resistance 
of  guinea-pigs  to  accidental  infection.  He  has  also  mentioned 
the  fact  that  the  animals  so  treated  suffer  a  considerable  loss  of 
weight.  It  has  been  noticed  in  the  present  experiments  that  this 
loss  of  weight  in   guinea-pigs   receiving   2   c.c.   intraperitoneally 


Immunological  Reactions  of  Oidiomycosis  195 

every  fourth  day  may  be  extreme,  the  animals  become  flabby  to 
the  touch  and  appear  markedly  cachectic.  They  slowly  recover 
when  the  injections  are  discontinued.  Bearing  in  mind  the  above 
findings,  it  became  of  interest  to  test  infected  animals  with  gradu- 
ated doses  of  the  extract.  The  results,  as  shown  in  the  following 
experiments,  appear  to  be  of  possible  importance. 

A  series  of  10  guinea-pigs  which  had  recovered  from  spotted 
fever  eight  months  to  a  year  before  were  given  intraperitoneal 
injections  of  o.i  gm.  of  oidiomycetes  from  an  agar  slant  culture 
on  January  21,  1909.  As  soon  as  nodules  became  palpable  in  the 
testicles,  five  of  these  pigs  were  given  subcutaneous  injections  of 
extract  ("BIR  1/19/09,"  CHCI3  preservative)  as  indicated  below, 
the  other  five  animals  being  used  as  controls. 

Guinea-pig  45:  January  21,  1909,  control  pig;  wt.  =  68o  gm.  o.i  gm.  intraperito- 
neally;  January  25,  nodules  palpable  in  testicles;  February  8,  nodules  in  testicles 
about  I  cm.  in  diameter;  ]\Iarch  i,  testicles  normal. 

Guinea-pig  46:  January  21,  1909,  control  pig;  wt.  =  775  gm.  0.1  gm.  intraperi- 
toneally.     Result  like  guinea-pig  45. 

Guinea-pig  47:  control  pig;  wt.  =  5i5  gm.;  January  25,  slight  induration  of  testicles; 
February  8,  same;   true  nodules  never  developed;   March  i,  testicles  normal. 

Guinea-pig  48:  control  pig;  wt.  =  5i5  gm.;  January  25,  slight  induration  of  testicles; 
February  8,  testicles  contain  nodules,  some  of  which  are  about  i  cm.  in  diameter; 
March  i,  testicles  normal. 

Guinea-pig  49:  control  pig;  wt.  =  955  gm.;  January  25,  small  nodules  can  be  felt  in 
the  testicles;  February  8,  large  nodules  are  palpable  in  the  testicles;  March  i, 
testicles  normal. 

Guinea-pig  50:  January  21,  1909,  wt.  =  69o  gm.  0.1  gm.  oidiomycetes  intraperi- 
toneally.  Received  subcutaneous  injection  of  0.5  c.c.  extract  January  25,  30, 
Februarj'  4,  9,  15,  23;  January  25,  nodules  palpable  in  testicles.  Small  nodule 
at  point  of  inoculation  drained  and  sterilized  with  95  per  cent  carbolic  acid  and 
absolute  alcohol;  wound  healed  rapidly.  Pus  contained  large  numbers  of  oidiomy- 
cetes. February  8,  small  nodules  in  testicles;  February  28,  animal  fully  recovered. 

Guinea-pig  51:  January  21,  1909,  wt.  =  68o  gm.  0.1  gm.  oidiomycetes  intraperi- 
toneally.  Received  0.5  c.c.  extract  subcutaneously  January  25,  30,  February 
4,  9,  15,  and  23;  January  25,  nodules  in  testicles;  February  8,  nodules  in  tes- 
ticles nearly  as  large  as  those  in  control  pigs;  March  i,  animal  fully  recovered. 

Guinea-pig  52:  January  21,  1909,  wt.  =  690  gm.  o.i  gm.  oidiomycetes  intraperi- 
toneally.  Received  0.5  c.c.  extract  subcutaneously  January  25,  27,  29,  Feb- 
ruary 1,3,  5,8,  lo,  12,  15,  17,  23,  and  25;  January  25,  slight  hardening  of  tip  of 
right  testicle  noted;  February  8,  nodules  in  testicles  fully  as  large  as  the  largest 
among  the  control  pigs;  February  25,  on  this  date  the  nodules  had  almost 
disappeared  so  that  the  injections  were  discontinued. 

Guinea-pig  53:  January  21,  1909,  wt.  =  72S  gm.  0.1  gm.  oidiomycetes  intraperi- 
toneally.     Received  0.5  c.c.  extract  subcutaneously  January   25,   27,  and   29; 


196  Contributions  to  Medical  Science 

January  25,  nodules  palpable  in  testicles;  February  i,  found  dead.  Autopsy: 
The  abdominal  parietes,  testicles,  seminal  vesicles,  and  diaphragm  are  peppered 
with  nodules  varying  in  diameter  from  0.5  mm.  to  4  mm.  A  few  similar  nodules 
appear  on  the  surfaces  of  the  spleen  and  liver.  In  the  great  omentum  along  the 
greater  curvature  of  the  stomach  is  a  thick  fibrinous  mass;  a  mass  of  cheesy  pus 
I  cm.  in  diameter  and  0.5  cm.  in  thickness  surrounded  by  a  hyperaemic  zone 
occurs  on  the  anterior  abdominal  wall  at  the  point  of  inoculation.  A  few  grayish 
patches,  i  to  3  mm.  in  greatest  width,  appear  beneath  the  epicardium  and  seem 
to  involve  the  heart  muscles.  The  lungs  do  not  appear  to  be  so  collapsible  as 
normal;  they  appear  to  be  congested;  no  gross  nodules.  Cultures  from  the 
various  nodules  show  oidiomycetes  in  pure  culture.  Histological  examination 
of  the  lungs  shows  that  there  is  no  edema  nor  e.xudation. 
Guinea-pig  54:  January  21,  1909,  wt.  =  6io  gm.  o.i  gm.  oidiomycetes  intraperi- 
toneally.  Received  0.5  c.c.  extract  subcutaneously  February  i,  3,  5,  8,  10,  and 
12;  February  i,  nodules  palpable  in  testicles;  February  8,  fairly  large  nodules 
in  testicles;  February  15,  found  dead.  Autopsy:  The  right  testicle  is  trans- 
formed into  a  sac  of  pus  containing  oidiomycetes  in  large  numbers.  Scattered 
nodules  i  to  5  mm.  in  diameter  are  found  on  liver,  spleen,  mesentery,  left  testicle, 
and  abdominal  wall.  A  nodule  in  the  intermuscular  fascia  of  the  anterior  abdom- 
inal wall  is  I  cm.  in  diameter  and  2  to  3  mm.  in  thickness,  and  contains  an  enor- 
mous number  of  budding  oidiomycetes.  The  left  lung  is  very  much  enlarged 
and  occupies  three-fourths  of  the  thoracic  cavity;  it  is  hard,  non-collapsible, 
dark  in  color,  with  hemorrhagic  spots.  Heart  and  right  lung  appear  normal. 
Material  from  various  nodules  smeared  on  glucose  agar  slants  yielded  oidiomy- 
cetes in  pure  culture.  Histologically  the  left  lung  appears  to  be  in  a  state  of 
pneumonic  consolidation. 

This  experiment  suggests  that  in  the  extract  we  may  have  a 
means  of  modifying  to  a  large  extent  the  course  of  an  oidiomycetic 
infection  in  guinea-pigs.  Those  animals  which  received  0.5  c.c. 
of  the  extract  subcutaneously  every  fifth  day  seemed  to  suffer  a 
milder  infection  than  the  control  pigs  and  to  make,  possibly,  a 
somewhat  more  speedy  recovery ;  of  those  animals  receiving  o .  5 
c.c.  extract  subcutaneously  every  second  day,  one— guinea-pig 
42 — paralleled  very  closely  the  control  pigs  in  this  infection; 
guinea-pig  53  died  in  11  days  of  generalized  oidiomycosis,  and 
guinea-pig  54  died  25  days  after  injection,  autopsy  showing  exten- 
sive oidiomycosis  of  the  abdominal  parietes  and  viscera  with  a 
terminal  lobar  pneumonia;  0.5  c.c.  of  the  extract  injected  sub- 
cutaneously every  second  day  into  infected  guinea-pigs  may  be 
toxic;  0.5  c.c.  of  the  extract  injected  subcutaneously  every  fifth 
day  into  similarly  infected  guinea-pigs  apparently  is  not  toxic  and 
seemingly  increases  the  animal's  resistance  to  the  disease.  Broad 
generalizations  from  a  single  experiment  such  as  this  are,  of  course, 


Immunological  Reactions  of  Oidiomycosis  197 

unwarranted,  but  it  seems  possible  that  further  work  along  the 
lines  indicated  by  these  results  may  lead  to  the  development  of 
a  technic  which  will  enable  us  to  treat  effectively  oidiomycetic 
infections. 

EXPERIMENTS   IN  ANAPHYLAXIS. 

In  testing  further  the  properties  of  the  "extract,"  an  attempt 
was  made  to  elicit  with  it  the  phenomenon  of  anaphylaxis  in 
guinea-pigs.  It  was  with  such  an  end  in  view  that  the  following 
experiments  were  performed.  The  temperature  of  the  animals 
was  observed  only  in  cases  where  specific  mention  of  that  fact  is 
made. 

Experiment  i. — Guinea-pig  i  received  an  intraperitoneal  injection  of  i  c.c.  of  "Extract 
BIH2,"  which  had  been  preserved  in  0.5  per  cent  phenol  for  about  13  months, 
on  October  27,  1908.  Sixteen  days  later  5  c.c.  of  the  "extract"  were  injected 
intraperitoneally.  Result:  no  convulsions  nor  tremors  of  any  kind;  pig  alive 
and  well  24  hours  later. 

Experiment  2. — Guinea-pig  17  was  treated  with  doses  of  "Extract  BIH2,"  similar  to 
those  given  guinea-pig  i.  An  interval  of  27  days  separated  the  two  injections. 
Results:  Negative,  similar  to  Experiment  i. 

Experiment  3. — Guinea-pig  17  received  an  intraperitoneal  injection  of  0.05  gm.  of 
organism  BIR  on  November  12,  1908.  Twelve  days  later  the  animal  was  found 
to  have  lost  90  gra.  in  weight;  both  testicles  had  become  swollen  and  hard. 
Five  c.c.  of  "Extract  BIH2"  were  injected  intraperitoneally.  There  ensued  a 
temporary  fall  of  about  8°  F.  in  temperature  which  returned  to  normal  within 
24  hours.     No  further  symptoms  appeared.     The  animal  thereafter  gained  weight. 

Experiment  4. — Guinea-pig  31  was  given  5  c.c.  of  centrifugated  "Extract  BIR" 
(preservative  0.5  per  cent  phenol)  intraperitoneally  on  January  4,  1909.  The 
injection  was  repeated  on  January  15,  without  the  production  of  symptoms. 

Experiment  5. — Guinea-pig  40  received  o .  2  c.c.  "  Ext.  BIR"  (preservative  o .  5  per  cent 
phenol)  intraperitoneally  on  January  15,  1909.  Guinea-pig  41  received  0.5 
c.c.  "Ext.  BIR"  intraperitoneally  on  January  15.  Guinea-pig  42  received  i.o 
c.c.  "Ext.  BIR"  intraperitoneally  on  January  15.  Guinea-pig  43  received  1.5 
c.c.  "Ext.  BIR"  intraperitoneally  on  January  15.  Guinea-pig  44  received  2.0 
c.c.  "Ext.  BIR"  intraperitoneally  on  January  15.  On  January  26 — interval  of 
1 1  days — guinea-pigs  40  and  43  received  5  c.c.  of  Ext.  BIR  intraperitoneally — 
no  reaction.  On  January  28  guinea-pig  41  and  guinea-pig  42  with  a  normal  con- 
trol pig  received  7  c.c.  Ext.  BIR  intraperitoneally.  Negative  results.  Guinea- 
pig  44  died  of  pneumonia  before  he  was  tested. 

Experiment  6. — Guinea-pig  55,  wt.  660  gm.,  received  o.i  c.c.  Ext.  BIR  (preserva- 
tive CHCLj  0.3  per  cent)  intraperitoneally  January  22,  1909.  Guinea-pig  56, 
wt.  795  gm.,  received  0.7  c.c.  Ext.  BIR  intraperitoneally.  Guinea-pig  57,  wt. 
830  gm.,  received  i .  5  c.c.  Ext.  BIR  intraperitoneally.  Guinea-pig  58,  wt.  680 gm., 
received  3.0  c.c.  Ext.  BIR  intraperitoneally.  After  an  interval  of  14  days, 
each  of  these  four  animals  and  each  of  two  normal  control  pigs  were  given  an 


198  Contributions  to  Medical  Science 

intraperitoneal  injection  of  5  c.c.  of  the  extract.  Only  one  animal  presented 
any  untoward  symptoms  and  one  of  the  control  animals  died  in  about  12  hours. 
Postmortem  examination  revealed  an  acute  fibrinous  peritonitis. 

Experiment  7. — Each  of  three  small  guinea-pigs  averaging  about  250-300  gm.  in 
weight  was  given  0.3  c.c.  of  Ext.  BIR  intraperitoneally  on  February  26,  1909. 
The  chloroform  used  as  a  preservative  had  been  allowed  to  evaporate  before  the 
extract  was  used.  After  an  interval  of  17  days  each  of  the  above  animals  and 
two  normal  pigs  of  similar  size  received  5  c.c.  of  chloroform  free  extract  intraperi- 
toneally. All  of  the  animals  appeared  to  be  sick  immediately  following  the 
injection.  The  three  sensitized  pigs  were  found  dead  next  morning.  One  of  the 
control  animals  died  a  week  later  without  apparent  cause;  the  other  remained 
in  normal  condition. 

Experiment  8. — A  couple  of  grams  of  moist  oidiomycetes  were  ground  in  the  porcelain- 
ball  mill  for  three  hours,  and  then  taken  up  with  NaCl  solution  (0.85  per  cent). 
Four  c.c.  of  this  fresh  extract  were  injected  into  each  of  two  medium  sized  guinea- 
pigs.  Twenty-six  days  later,  each  of  these  two  animals  and  two  normal  pigs  were 
given  10  c.c.  of  a  fresh  extract  intraperitoneally.  The  two  sensitized  animals  were 
foimd  dead  the  following  morning;  one  control  pig  died  two  days  later,  the  other 
a  week  later.  This  extract  yielded  the  usual  white,  flocculent  precipitate  upon 
the  addition  of  absolute  alcohol. 

Experiment  g. — Fresh  moist  organisms  were  ground  in  a  mortar  with  sterile  sand  in 
0.85  per  cent  NaCl  solution  containing  0.2  per  cent  of  normal  NaOH.  The 
total  volume  of  the  ground  material  was  made  up  to  25  c.c.  by  the  addition  of 
the  salt  solution  containing  o.  2  per  cent  of  normal  NaOH.  Five  c.c.  of  the  total 
was  sediment.  The  fluid,  when  injected,  was  yellowish  white  and  opaque. 
Guinea-pig  (a):  wt.  140  gm.,  received  o.i  c.c.  intraperitoneally.  Guinea-pig 
(i):  wt.  215  gm.,  received  0.3  c.c.  intraperitoneally.  Guinea-pig  (c):  wt.  225 
gm.,  received  0.5  c.c.  intraperitoneally. 
Eighteen  days  later  guinea-pig  (6)  and  a  normal  guinea-pig  of  similar  size  were 

given  an  intraperitoneal  injection  of  the  original  fresh  extract,  which  had  been  kept 

in  the  ice-box,  warmed  to  37°  C.     The  extract  at  this  time  yielded  the  usual  precipi- 
tate with  absolute  alcohol. 

Immime  Pig  (6):  Temp,  before  injection,  10:30  a.m.,  102° F.    Injection  10:50  a.m. 

Normal  Pig:  "  "  "  "         "      100.6°  F.       " 

Immune  Pig  (6):  Temp.  10:55,  100. 2° F. 

Normal  Pig:  "  "  99.8°F. 


12:1s 

1:4s 

2:45 

3:4s 

4:4s 

5:43 

10:30  A.M. 

99.8°  F. 

98. 2° F. 

97°      F. 

97.6°  F. 

98°     F. 

99. 2° F. 

102. 6°F. 

96.8°  F. 

97.6°  F. 

96. 8°  F. 

97-3°F. 

98. 2°  F. 

98.i°F. 

100. 6°  F. 

Two  days  after  this,  an  interval  of  20  days  after  sensitization,  guinea-pigs  (a)  and 
(c)  and  each  of  two  control  animals  of  a  size  approximating  that  of  pigs  (o)  and  (c) 
were  given  intraperitoneally  5  c.c.  of  a  freshly  prepared  extract,  warmed  to  37°  C, 
similar  to  that  originally  employed.     Injection  at  12:20  p.m. 

Temp,  at    11:50  12:4s  1:50  2:30 

Guinea-Pig  (a) :   102°      F.     ioi.4°F.       98.8°  F.       944°  F. 
Control:  100.4°  F.       94°      F.       96.4°  F.       95.4°  F. 

Guinea-Pig  (c):    ioi.o°F.     100. 4°F.       97.8°F.       94.2°F. 
Control:  101°     F.     100. 2°  F.      96.8°  F.      94.6°  F. 


4:3s 

11:00  A.M. 

96.4°  F. 

102°     F. 

96°      F. 

102°     F. 

94°      F. 

104°     F. 

94.4°  F. 

100.8° F. 

Immunological  Reactions  of  Oidiomycosis 


199 


In  addition  to  the  fall  in  temperature,  pigs  (6)  and  (c),  and,  to  a  lesser  extent,  (a) , 
developed  a  tense,  swollen,  and  apparently  painful  abdomen  which  subsided  as  the 
temperature  rose  to  normal.  There  was  no  suggestion  of  such  a  condition  among 
the  control  pigs. 

From  the  above  experiments,  the  conclusions  seem  justified 
that:  (i)  The  extract  is  toxic  ahke  for  normal,  sensitized,  and 
infected  animals  as  indicated  by  the  sharp  fall  in  temperature 
following  intraperitoneal  injection  of  considerable  quantities. 
(2)  It  seems  possible  to  demonstrate  the  phenomenon  of  anaphy- 
laxis by  the  use  of  freshly  prepared  extract,  or  of  extract  freed 
from  its  preservative  (CHCI3) ,  the  reaction  manifesting  itself  either 
by  the  death  of  the  animal  within  1 2  to  20  hours,  or  in  the  develop- 
ment of  a  sharp  intraperitoneal  reaction  within  two  or  three  hours 
following  the  intoxicating  injection,  which  subsides  within  24 
hours.  Large  doses  may  kill  the  control  animals;  in  such  cases, 
the  death  of  the  sensitized  animals  usually  precedes  that  of  the 
normal  animals. 


passive  immunization. 

In  view  of  the  seemingly  low  grade  of  active  immunity  which 
guinea-pigs  developed  against  oidiomycetes,  it  appeared  that 
efforts  to  confer  a  passive  immunity  could  be  more  profitably 
expended  along  other  lines  so  that  no  such  experiments  were 
undertaken.  In  Dr.  H.  T.  Ricketts'  notes  on  his  work  on  oidio- 
mycosis, which  he  kindly  placed  at  my  disposal,  the  following 
experiment  is  reported: 

Rabbit  44,  was  immunized  against  BIR  as  follows: 
November  19,  1905,  i  tube  of  glucose-agar  culture  (killed)  subcutaneously.     Local 

abscess  produced. 
November  26,  1905,  i    tube  of  glucose-agar  culture  (killed)  intraperitoneally. 
December     4,  1905,  i§ 
December   12,  1905,  15 
December  19,  1905,  i^ 
December   26,  1905,  15 
January         7,  1906,  2 
January       17,  1906,  2 
January       23,  1906,  i    gram  living  agar  culture  intraperitoneally. 
February      9,  1906,  i       "         "        "  "  " 

February     16,  1906,  12  c.c.  of  blood  drawn.     This  blood  was  defibrinated,  centrifu- 

gated,  and  the  serum  used  in  the  following  experiment: 


2CX> 


Contributions  to  Medical  Science 


Experiment  lo. — February  i6,  1906.  Weighed  amounts  of  moist  agar  culture  were 
mixed  with  varying  quantities  of  "immune"  rabbit  serum  and  injected  into  the 
peritoneal  cavity  of  a  series  of  guinea-pigs.  Control  experiments  were  carried 
out  with  normal  rabbit  serum  and  with  the  untreated  organisms. 

TABLE  3. 


Date 


Wt.  in  Grains 


Result 


Guinea-pig  i  received  0.3  gm. 
Bl-l-2  c.c.  im.  serum,  intraperi- 
toneally  on  2/16. 


Guinea-pig  2  received  0.3  gm. 
Bl-t-i  c.c.  im.  ser.-fi  c.c.  NaCl 
sol.  (0.85  per  cent)  intraperitone- 
ally  on  2/16. 

Guinea-pig  3  received  0.3  gm. 
Bl-(-o.s  c.c.  im.  ser.-f-i.s  c.c. 
NaCl  sol.  (0.8s  per  cent)  intra- 
peritoneally  on  2/16. 

Guinea-pig  4  received  0.3  gm. 
Bl-j-2  c.c.  normal  serum,  intra- 
peritoneaJly  on  2/16. 

Guinea-pig  s  received  0.3  gm. 
Bl-f  I  c.c.  normal  serum-f-i  c.c. 
NaCl  sol.  (0.85  per  cent)  intra- 
peritoneally  on  2/16. 

Guinea-pig  6  received  0.3  gm. 
Bl-l-0.5  c.c.  normal  serum -|- 
i.S  c.c.  NaCl  sol.  (0.85  per  cent) 
intraperitoneally  on  2/16. 

Guinea-pig  7  received  0.3  gm. 
BH-2  c.c.  NaCl  sol.  (0.85  per 
cent)  intraperitoneally  2/16. 


Guinea-pig  8  received  dose  similar 
to  that  of  guinea-pig  7. 


2/16/06 
3/  7/06 
3/16/06 
3/24/06 

2/16/06 
3/  7/06 
3/16/06 
3/24/06 

2/16/06 
3/  7/06 
3/16/06 
3/23/06 

2/16/06 
3/  7/06 
3/1 1/06 

2/16/06 
3/  7/06 
3/18/06 


2/16/06 
3/  7/06 
3/16/06 


2/16/06 
3/  7/06 
3/16/06 
3/29/06 

2/16/06 


390 
39S 
360 
270 

37S 
370 
340 
24s 

378 

3SO 

28s 

? 

400 

400 

? 

400 
375 
270 


42s 
410 
380 


386 
36s 
32s 
230 

390 


Result:  Death  on  3/24  from  gen- 
eralized oidiomycosis;  interval 
of  36  days.  Total  loss  of  wt.  = 
120  gm. 

Result:  Death  on  3/24  from  gen- 
eralized oidiomycosis;  interval 
of  36  days.  Total  loss  of  weight 
=  130  gm. 

Result:  Death  on  3/23,  from  gen- 
eralized oidiomycosis;  interval 
of  35  days. 


Result:  Death  on  3/11,  from  gen- 
eralized oidiomycosis;  interval  of 
23  days. 

Result:  Death  on  3/18  from  gen- 
eralized oidiomycosis;  interval 
of  30  days.  Total  loss  of  weight 
=  130  gm. 

Result:   Animal  lost. 


Result:  Death  from  generalized 
oidiomycosis  on  3/29.  Interval 
of  41  days.  Total  loss  of  weight 
=  156  gm. 

Result:  Accidentally  killed  2/22/06, 
six  days  after  injection. 


One  cannot  argue  from  these  results  that  the  "immune"  serum 
conferred  an  immunity  of  any  degree.  It  should  be  remembered 
in  this  connection,  however,  that  rabbit  serum  is  somewhat  toxic 
for  guinea-pig  leukocytes,  a  property  which  might  mask  the  pro- 
tective power  of  such  a  serum.  There  seems  to  be  a  suggestion 
of  a  reaction  of  this  kind  in  the  results  of  the  above  experiment, 
but  more  work  must  be  done  before  conclusions  may  be  drawn. 


immunological  reactions. 
Some  preliminary  work  along  this  Kne,  carried  out  with  the 
organism  "BIR,"  was  done  in  1905  by  Dr.  H.  T.  Ricketts,  and 
continued  during  the  year  1906-7  by  Dr.  H.  E.  Eggers.  Dr. 
Ricketts  performed  the  following  experiment:  A  guinea-pig  was 
immunized  against  the  oidiomycetes  by  the  repeated  injection  of 


Immunological  Reactions  of  Oidiomycosis 


20I 


killed  organisms  and  of  extract  as  is  shown  in  the  accompanying 
table. 

Guinea-Pig   i. 

Inoculated  with 
o.  I  gm.  killed  agar  culture  intraperitoneally 

O.I      " 

O.I     "        "        "  "  " 

2  c.c.  of  extract  subcutaneously 


Killed  on  account  of  loss  of  weight,  and  serum  used 
for  precipitation  tests. 

Precipitation  Tests.    April  4,  1906.    Blastomyces  R — Extract  II. 
Serum  from  Immunized  Guinea-Pig  i. 

I.  Original  Extract  II,  after  prolonged  centrifugation  to  remove  all  sediment, 
was  used  as  antigen.    The  fluid  was  whitish-opalescent. 


Date 

Weight 

February    7 

February  16 

February  27 

March        9 

March       16 

390  gm 

March      30 

31S     " 

Extract 

Immune 
Serum 

Normal 
Serum 

NaCI  (0.8s 

per  cent) 

Sol. 

Result 

o.S  c.c. 
o.S  " 
"S  ! 
o.S 
o.S  " 
0.5    ' 

0.1  c.c. 
0.2   " 
0.3   " 
0.0  " 
0.0  " 
0.0  " 

0.0  c.c. 
0.0  " 
0.0  " 
0.1   " 
0.2   " 
0.3   " 

0.4  c.c. 
0.3   " 
0.2 
0.4  " 
0.3   " 
0.2 

Distinct    precipitate 
Marked            " 
Fairly  heavy    " 
No 

II.  Absolute  alcohol  precipitate  of  Extract  II,  freed  of  alcohol  and  redissolved 
in  NaCl  (0.85  per  cent)  solution.  Fluid  was  slightly  opalescent  but  much  less  so 
than  the  centrifugated  extract. 


Redissolved 
Precipitate 

Immune 
Serum 

Normal 
Serum 

NaCl  Sol. 

(o.8s  per 

cent) 

Result 

o.S  c.c. 
o.S   " 
o.S 
o.S   " 
OS   " 
o.S 

0.1  c.c. 
0.2   " 
0.3   " 
0.0  " 
0.0  " 
0.0  " 

0.0  c.c. 
0.0  " 
0.0  " 
0.1   " 
0.2   " 
0.3   " 

0.4  c.c. 
0.3   " 
0.2   " 
0.4  " 
0-3    " 
0.2    " 

Distinct    precipitate 
Marked            " 
Fairly  heavy    " 

No              •; 

III.  Clear,  colorless,  non-opalescent  filtrate  of  Extract  II  used  as  precipitogen. 


Filtrate 

Immune 
Serum 

Normal 
Serum 

NaCl  Sol. 

(0.8s  per 

cent) 

Result 

o.S  c.c. 
o.S   " 

o.S 

0.5 

0.1  c 

0.2 

0.3 

0.0 

0.0 

0.0 

.c. 

0.0  c.c. 
0.0  " 
0.0  " 
0.1   " 
0.2  " 
0.3   " 

0.4  c.c. 
0.3   " 
0.2   " 

0.4  " 
0.3  " 
0.2  " 

No  precipitate 
V'ery  faint  precipitate 
Small,  but  distinct  precip. 
No  precipitate 

Normal  serum  alone = no  precipitate. 
Immune  serum  alone  =  no  precipitate. 
Centrifugated  extract  alone  =  no  precipitate. 
Redissolved  alcoholic  precipitate  alone  =  no  precipitate. 
Filtrate  alone  =  no  precipitate. 


202  Contributions  to  Medical  Science 

The  records  of  work  done  by  Dr.  H.  E.  Eggers  were  lost  before 
the  present  work  was  begun.  Dr.  Eggers  has,  however,  written 
a  summary  of  his  work  from  memory  which  is  introduced  at  this 
point  with  his  permission : 

The  following  work  on  attempted  immunization  to  blastomycosis  was  carried  on, 
on  guinea-pigs,  rabbits,  and  goats. 

The  plan  was  followed  of  injecting  into  the  animals,  at  periods  of  from  seven  to 
eight  days,  an  extract  prepared  from  the  dried  blastomyces  by  grinding.  Before 
injection  care  was  taken  to  shake  the  material  thoroughly  to  secure  suspension  of  the 
solid  fragments  of  the  organisms. 

Using  this  suspension,  work  was  begun  on  12  guinea-pigs  and  9  rabbits.  0.5 
c.c.  of  the  material  was  injected  into  the  guinea-pigs,  i  c.c.  into  the  rabbits.  Injec- 
tions were  for  the  most  part  intraperitoneal;  they  were  repeated  every  seven  to  eight 
days.  The  condition  of  the  animals  was  controlled  by  weighing.  If  any  considerable 
loss  of  weight  followed  an  injection,  one,  occasionally  more,  periods  were  passed  over 
without  injection.  In  the  case  of  some  of  the  animals  a  sharp  reaction  followed  the 
first  few  injections;  several  died  within  two  or  three  days.  Postmortem  examina- 
tion revealed  acute  parenchymatous  changes,  particularly  in  the  liver  and  kidneys. 
Other  animals  were  substituted  for  the  ones  so  lost.  Several  of  the  rabbits  and  guinea- 
pigs  became  pregnant  during  the  course  of  the  work;  such  animals  were  injected 
subcutaneously  while  pregnant. 

These  injections  were  carried  on  from  early  in  October,  1906,  until  January,  1907. 
At  this  time  serum  from  several  of  the  rabbits  was  tested  with  the  homologous  extract 
for  a  precipitation  reaction.  A  positive  reaction,  not  given  beyond  a  dilution  of 
1 :  20,  was  found  at  this  time  with  one  animal.  The  serum  of  another  one,  which  had 
shortly  previously  given  birth  to  young,  as  well  as  the  serum  of  the  young  was  tested, 
both  being  negative.  With  the  animal  showing  the  slight  positive  serum  reaction 
the  injections  were  kept  up  for  a  month;  no  increase  in  reaction  resulted,  and  shortly 
after  this  the  rabbit  died  of  a  meningitis. 

The  periodic  injection  was  carried  on  longer,  the  doses  being  increased  until  the 
guinea-pigs  received  i  c.c.  at  a  time  and  the  rabbits  two.  Evidently  their  vitality 
was  considerably  lowered  by  the  repeated  injections,  as  a  considerable  number,  of 
the  rabbits  especially,  died  of  secondary  infections — meningeal  for  the  most  part. 
Although  the  injections  of  the  few  remaining  rabbits  were  continued  until  June,  1907, 
in  no  case  was  I  successful  in  getting  a  precipitation  reaction  beyond  a  dilution  of 
1:20.  The  rabbits  showing  even  this  reaction  were  too  few  to  enable  any  decisive 
test  as  to  their  degree  of  immunization  to  be  made. 

Early  in  April,  1907,  that  is,  six  months  after  injection  had  been  begun,  six  of  the 
eight  guinea-pigs  that  had  been  injected  a  sufiicient  number  of  times  to  warrant 
e.xpectation  of  results  were  injected  intraperitoneally  with  varying  doses  of  a  freshly 
prepared  suspension  of  blastomyces,  the  doses  being  0.25,  0.5,  i,  2,  3,  and  4  c.c. 
respectively.  Six  normal  animals  were  injected  with  similar  doses.  In  no  case  did 
any  of  the  animals  die  with  any  of  the  findings  of  blastomycosis.  The  general  lowered 
resistance  of  the  previously  injected  animals  was  betrayed  by  the  fact  that  for  the 
most  part  they  were  survived  by  the  normal  animals. 

Late  in  October,  1906,  weekly  injections  of  a  goat  were  begun,  the  animal  receiving 
at  the  start  3  c.c.  of  the  extract  subcutaneously,  later  increased  gradually  to  5  c.c. 


Immunological  Reactions  of  Oidiomycosis  203 

The  serum  of  this  animal,  tested  late  in  January,  was  found  to  give  a  positive  precipitin 
reaction  with  the  homologous  extract  in  a  dilution  of  i :  25.  With  the  extract  of 
another  strain  of  the  organisms — "H  II" — it  gave  a  precipitate  in  a  dilution  of  1:12.5; 
with  the  third  strain  no  reaction  at  all  occurred.  Injections  were  continued,  until 
the  animal  was  noticed  to  be  becoming  thin  and  feeble.  It  finally  succumbed  late  in 
February;  postmortem  examination  revealed  a  bronchopneumonia,  other  tissues 
negative.  The  serum  at  the  time  of  its  death  reacted  in  the  same  manner  and  in 
similar  dilutions  to  the  results  just  given. 

Work  was  at  once  begun  with  another  goat,  the  injections,  of  3  c.c.  each,  being 
made  subcutaneously  at  eight  day  intervals.  This  was  kept  up  until  the  beginning 
of  June,  three  months,  at  which  time  precipitin  reactions  similar  in  every  way  to  those 
obtained  with  the  preceding  animal  were  found. 

As  the  formation  of  antibodies  with  this  method  of  repeated  small  injections  was 
evidently  rather  slight,  a  second  goat  was  obtained,  and  injected  with  larger  and 
ascending  doses  at  somewhat  greater  intervals,  10  or  11  days.  The  first  dose,  of  20 
c.c.  of  extract,  given  subcutaneously,  produced  a  marked  reaction,  the  animal 
being  quite  sick  for  three  or  four  days  afterward.  Local  reaction  at  the  site  of  injec- 
tion was  extremely  slight.  The  next  dose  of  30  c.c.  was  again  followed  by  a  marked 
reaction,  the  third  dose  of  40  c.c.  by  considerably  less. 

The  same  plan  was  followed  on  rabbits  and  guinea-pigs.  Before  this  time  the 
writer  left  Chicago  for  the  summer,  and  the  injections  were  made  without  the  posses- 
sion of  facilities  for  testing  the  serum  reactions  of  the  animals.  The  change  was 
disastrous  to  the  rabbits,  as  all  of  them  died  of  an  acute  enteric  affection.  The  quantity 
of  extract  on  hand  did  not  warrant  work  being  begun  on  new  rabbits.  The  guinea- 
pigs  were  injected  successively  with  doses  of  i,  3,  5,  and  7  c.c.  After  two  doses  of 
this  last  amount  had  been  given,  it  was  dropped  back  to  2  c.c.  per  dose;  the  dosage 
of  the  goat,  after  reaching  40  c.c,  was  dropped  back  to  10.  In  the  case  of  the  guinea- 
pigs,  the  reaction  from  the  larger  doses  used  here  was  no  more  marked  than  from  all 
the  first  few  smaller  doses  used  previously. 

^\^lile  out  of  the  city,  the  first  of  the  two  goats,  which  was  still  being  injected 
with  the  repeated  small  doses  of  extract,  died;  postmortem  examination  showed  a 
condition  of  apparently  long-continued  pylorospasm,  caused  by  the  lodging  of  a  pin 
in  the  gastric  mucosa. 

The  remaining  animals,  the  second  goat  and  the  guinea-pigs,  were  brought  back 
to  the  city.     At  this  time  the  writer  was  obliged  to  discontinue  the  work. 

In  the  fall  and  winter  of  1908-9  the  work  on  precipitins  was 
continued,  guinea-pigs  being  used  as  the  experimental  animals. 
The  results  are  tabulated  below.  Some  of  the  animals  were  immu- 
nized by  injections  of  the  extract  of  another  organism  than  BIR, 
known  in  the  laboratory  as  Bl  H  II. 

The  experiment  of  Dr.  Ricketts  with  the  centrifugated,  filtered, 
and  redissolved  alcoholic  precipitate  of  the  extract  was  repeated, 
using  the  serum  of  guinea-pig  5  as  the  immune  serum,  with  positive 
results  in  all  cases.  Thereafter  the  centrifugated  extract  was  used 
exclusively  as  the  precipitogen  in  the  precipitation  experiments. 


204  Contributions  to  Medical  Science 

The  technic  used  in  the  work  was  as  follows:  Blood  was  drawn, 
usually  from  the  guinea-pig's  heart,  defibrinated,  and  centrifugated, 
and  varying  quantities  of  the  clear  serum  added  to  a  constant 
amount  of  centrifugated  extract  in  small  test-tubes,  and  the  mix- 
tures made  up  to  a  uniform  volume  by  the  addition  of  NaCl  solu- 
tion (0.85  per  cent).  The  tubes  were  then  incubated  at  37°  C. 
for  two  hours,  placed  in  the  ice-chest,  and  results  observed  after 
24  hours.  Control  tubes  of  normal  serum  plus  extract  in  dilutions 
similar  to  those  of  the  immune  serum,  and  of  immune  serum  and 
NaCl  solution,  normal  serum  and  NaCl  solution,  and  extract  plus 
NaCl  solution,  were  always  prepared  as  a  part  of  each  experiment. 
It  was  noticed  that  occasionally  after  72  to  96  hours  in  the  ice- 
chest  precipitates  formed  in  tubes,  which  at  the  end  of  24  hours 
were  negative.  As  the  control  tubes  remained  clear  and  without 
sediment,  and  as  no  proofs  of  contamination  could  be  obtained  by 
cultural  methods  or  by  direct  microscopical  examination,  it  was 
assumed  that  such  precipitates  probably  represented  a  specific 
reaction  and  they  were  therefore  noted  in  subsequent  experiments. 
Once  in  a  while,  the  serum  of  normal  animals  formed  a  precipitate 
with  the  extract.  Of  the  22  normal  animals  tested,  2  or  approxi- 
mately 10  per  cent  gave  positive  reactions,  one  in  a  dilution  of  1 13 
after  24  hours  and  the  other  in  a  dilution  of  i :  10  on  the  4th  day. 

In  Table  4  the  figures  1:7,  1:10,  etc.,  represent  the  highest 
dilutions  at  which  precipitates  appeared;  a  dash  ( — )  indicates 
that  no  observations  were  made  and  "o"  indicates  absence  of 
precipitate. 

Six  animals  were  tested  more  than  once.  Their  history  appears 
in  Table  5. 

Of  the  32  immune  sera  tested,  22  reacted  positively  in  from  24 
to  96  hours.  Nine  were  absolutely  negative  in  so  far  as  they  were 
observed.  Of  the  sera  of  those  animals  which  were  immunized  by 
the  injection  of  extract  alone,  8  reacted  positively  in  24  hours, 
o  in  48  hours,  7  in  72-96  hours  in  one  or  another  of  the  tests,  and  4 
were  negative;  of  the  sera  of  those  which  received  the  organisms 
alone,  4  reacted  positively  in  24  hours,  o  in  48  hours,  i  in  72-96 
hours,  and  4  were  negative ;  of  the  sera  of  the  animals  which  received 
both  extract  and  organisms,  2  were  positive  in  24  hours  and  i  was 


Immunological  Reactions  of  Oidiomycosis 


205 


TABLE  4. 
Precipitins. 


GUINZA- 
PlG  No. 

No.  Injec- 
tions OF 

No.  OF 

Days 

Inter- 
val 
Since 

Average 
Dose 

No.  In- 
jection 
Living 
Organ- 
isms 

Dose 
Gm. 

No.  OF 
Days  In- 
terval 
Since  Re- 
covery 
FROM  Last 
Injection 

Results 

Ext. 
HII 

Ext. 
BIR 

Last          c.c. 

Injec-  ! 
tion 

24 
Hrs. 

48 
Hrs. 

72-96 
Hrs. 

I 

s* 

8 

10 

I 

17 

23 

31 

32 

36 

39 

43 

49 

50 

&:::::: 

S6 

S7 

S8 

7S 

83 

9a 

9S 

II 

Ill 

0 

4 

9 

5 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

0 

0 

0 
0 

0 
0 

4 
0 

8 

0 
0 
0 
0 
2 
S 
8 
4 
9 
0 
6 
6 
13 
9 

0 

0 

0 
0 

2 

2 

4 
37 

13 
28 

24 
6 
14 
11 
IS 

119 
122 

'11 
48 

48 

28 
2 

2 

(  H II  I 
i   BIR  2 

2 

s 

2 
2 
2 
2 

S  subcu. 
S  subcu. 
5  subcu. 

3 

3 

3 

ist  dose 
20  c.c. 
(  2d  10  c.c. 

0 
0 

0 

0 

3 

I 
I 
0 
0 
0 
0 

2 

I 
I 
I 
0 
0 
0 

2 

S 

3 
3 
0 

0 

O.I 

o.os 
0.01 

aver,  o.i 

O.I 
O.I 
O.I 

(  1st  dose 

(     2d  O.I 

(2d  O.s 
<  Others  O.I 
(      each 
aver.  0 .  i 

28 

about  26 

20 

about  19 

119 

122 
about  133 

about  60 
about  60 

0 
1:20 

1:20 

0 

0 

0 

0 

0 

0 

0 

0 

1:20 

0 

1:7 

i:S 

0 

0 
0 

0 

0 

ti:io 
Ji:lo 

0 
0 

0 
0 
0 

0 
1:10 
1:10 

0 

0 
0 

0 

0 
1:20 
1:30 

t:30 

1:20 

1:20 

?(o) 

1:7 

1:10 

1:10 

0 
0 

0 
i:  10 

*  Not  tested  in  higher  dilution. 
t  Ext.  Hi  used  as  precipitogen. 


t  Blood  drawn  July  13  but  not  tested  until  July  22. 


TABLE  s. 


GtJINEA 

Pig 
No. 

Total  No.  Injections  before 

No.  OF 

Days  Interval 

BETWEEN  Time  of 

RF-SITLTS 

ist  Test 

2d  Test 

3d  Test 

Last  Injection  or 
Infection  and 

Ext. 

Liv. 
Orgs. 

Ext. 

Liv. 
Orgs. 

Ext. 

Liv. 
Orgs. 

ISt 

Test 

3d 

Test 

Test 

ist 
Test 

2d 

Test 

■fL 

13*.... 
3S 

37 

38 

55 

81 

17 
8 

8 

8 
12 

0 

0 
0 

0 

0 
0 

4 

23 
14 

14 

II 
12 

0 

0 

0 

0 

0 
0 

4 

23 

3 

13 

14 

11 

13 
6 

27 

3 
6 

S 

3 
60 

60 

100 

1:20 

1:20 

after  3 

da. 

1:20 

0 
1:20 

1:20 

1:30 

I : 5  after 
4  da. 

I :  I  only 
after  4 

days 

1:20 
1:5  af- 
ter 48 

hrs. 

1:10 

0 

'At 


cavity. 


autopsy  of  guinea-pig  13  (date  of  3d  test)  oldiomycetes  were  found  in  a  nodule  in  the  abdominal 
Cultures  were  negative. 


2o6  Contributions  to  Medical  Science 

negative.  There  does  not  seem  to  be  any  very  constant  relation 
between  the  number  of  injections,  the  interval  between  the  time 
of  the  last  injection  and  that  of  the  test,  and  the  results  obtained; 
nor  does  the  quantity  of  the  living  organisms  introduced  seem  to 
be  of  paramount  importance.  About  all  that  one  seems  justified 
in  concluding  from  the  above  experiments  is  (i)  that  the  serum 
of  some  normal  guinea-pigs  will  cause  a  precipitate  when  mixed 
in  proper  proportions  with  oidiomycetic  extract,  and  (2)  that 
immunization  with  the  extract,  infection  with  living  organisms, 
or  combined  infection  with  oidiomycetes  and  immunization  with 
extracts  results  in  the  development  of  precipitating  substances 
in  the  serum  of  about  70  per  cent  of  the  animals  so  treated  (the 
percentages  in  the  above  experiments  were:  extract  alone  79  per 
cent,  organisms  alone  50  per  cent,  organisms  plus  extract  [small 
quantities  were  used]  67  per  cent).  These  figures  suggest  also 
that  Hving  organisms  plus  small  quantities  of  extract,  too  small  to 
produce  noticeable  symptoms  themselves,  may  be  much  more 
effective  in  calling  forth  antibodies  than  hving  organisms  alone 
and  so  rather  tend  to  support  the  deductions  made  from  the 
results  of  the  experiment  described  on  p.  204,  according  to  which 
it  was  suggested  that  the  extract  may  have  some  value  as  a  curative 
agent.  As  a  diagnostic  procedure,  with  guinea-pigs  at  any  rate, 
the  precipitation  reaction  would  have  to  be  controlled  by  symp- 
tomatological  findings;  a  negative  reaction  would  mean  nothing, 
a  positive  reaction  would  be  suggestive  but  not  conclusive  because 
of  the  fact  noted  above  that  normal  guinea-pig  serum  may  pre- 
cipitate with  the  extract  in  dilution  as  high  as  i :  10,  after  96  hours 
in  the  ice-chest,  at  least. 

According  to  Dr.  Eggers'  work,  the  results  with  rabbits  are 
even  more  variable  than  with  guinea-pigs.  The  goat  seems  to 
give  a  more  constant  reaction,  but  even  in  that  case  the  highest 
dilution  at  which  a  precipitate  formed  was  1:25,  which  is  cer- 
tainly not  evidence  of  a  very  powerful  serum. 

agglutinins. 

At  the  same  time  that  the  precipitating  powers  of  the  various 
sera  were  being  tested,  parallel  experiments  on  their  agglutinating 


Immunological  Reactions  of  Oidiomycosis 


207 


Serum 

Emulsion 

0.05  C.C. 

0.5  C.C. 

O.I       " 

O-S    " 

O.IS    " 

O-S    " 

0.2       " 

O.S    " 

0.25    " 

O-S   " 

0-3     " 

o-S   " 

0.3s   " 

o-S   " 

0.4     " 

o-S   " 

0.4s   " 

o-S   " 

o-S     " 

0.5   " 

I.O       " 

I .  loop 

power  were  attempted.  Emulsions  of  the  whole  organism  in 
0.85  per  cent  NaCl  were  used  in  these  experiments.  The  serum 
dilutions  were  as  follows:  For  methods  (a)  and  (6) — see  below — 
whole  serum  i  part  +  emulsion  i  part ;  50  per  cent  serum  i  part  + 
emulsion  i  part;  10  per  cent  serum  i  part  +  emulsion  i  part. 
For  method  (c)  the  dilutions  were : 

NaCl 
0.4s  C.C. 

0.4    " 

0.35  " 

0.3    " 
0.2s  " 

O.  2        " 

0.15     " 
O.I        " 

0.05  " 

0.0      " 
loop  of  moist  organisms       0.0      " 

Three  different  methods  were  tried:  (a)  hanging  drop  in  a  hollow- 
ground  slide,  (b)  quantities  of  i  to  2  c.c.  in  watch  glasses,  which 
could  be  observed  with  ease  both  with  the  unaided  eye  and  with 
the  microscope,  and  (c)  the  materials  were  placed  in  narrow  test- 
tubes  with  the  idea  that  something  might  be  learned  from  possible 
variations  in  the  rate  with  which  the  oidiomycetes  settled  out  of 
the  suspension.  In  each  experiment  the  following  mixtures  were 
made:  (i)  immune  serum  plus  emulsion,  (2)  normal  serum  plus 
emulsion,  (3)  salt  solution  plus  emulsion.  These  mixtures  were 
incubated  at  37°  C.  for  from  i  to  12  hours  and  then  allowed  to 
stand  at  room  temperature  until  observations  were  discontinued. 

The  results  were  practically  identical  with  the  normal  and 
immune  sera  in  all  the  experiments.  It  was  found  that  if  the 
organisms  were  allowed  to  stand  in  contact  with  the  serum,  normal  or 
immune,  for  a  few  hours,  a  beautiful  agglutination  reaction,  as 
judged  by  the  naked  eye,  occurred.  This  is  especially  noticeable 
in  the  watch-glass  preparations.  Under  the  microscope,  however, 
the  clumps  which  had  attracted  the  attention  resolved  themselves 
not  into  masses  of  agglutinated  oidiomycetes,  but  into  groups  of 
oidiomycetes  which  had  developed  h^phae,  and  it  was  to  the  appear- 
ance of  this  mycelial  network  that  the  apparent  agglutination 
was  due. 


2o8  Contributions  to  Medical  Science 

Regarding  the  rate  at  which  the  organisms  settled  out  of  sus- 
pension in  the  tubes,  no  differences  between  the  normal  and 
immune  sera  could  be  determined.  The  salt-solution  tube  sedi- 
mented  first,  the  other  tubes  sedimented  at  about  the  same  rate, 
and  the  organisms,  like  those  used  in  the  other  methods,  sprouted 
in  a  few  hours. 

It  is  true  that  sometimes,  especially  with  the  hanging-drop 
and  the  watch-glass  methods,  there  appeared  to  be  a  tendency  for 
the  organisms  to  gather  into  a  few,  large,  loosely  constructed 
groups,  but  this  occurred  with  normal  serum  as  well  as  immune, 
and  almost  as  frequently  with  the  salt  solution  alone,  and  the 
question  arose  whether  it  might  not  be  the  expression  of  an  imper- 
fect trituration  of  the  organism  in  the  preparation  of  the  emulsion. 
Certainly  no  specific  agglutination  of  oidiomycetes  by  "immune" 
sera  which  did  or  did  not  yield  a  precipitate  with  the  extract  was 
observed. 

Possibly  the  experiment  might  be  more  successful  if  the  organ- 
isms were  first  partially  ground  and  if  the  fragments  thus  obtained 
were  used;  the  spherules  described  within  the  conidia  and  the 
hyphae  might  also  be  used. 

LYTIC   AND    bactericidal   SUBSTANCES. 

It  had  been  observed  in  the  agglutination  experiments  that 
oidiomycetes  were  able  to  develop  hyphae  when  suspended  in 
undiluted  or  diluted  normal  and  immune  serum,  and  that  some- 
times organisms  which  had  been  subjected  to  the  action  of  such 
fluids  for  three  or  four  days  grew  when  planted  on  agar.  It  was 
therefore  argued  that  neither  normal  nor  immune  sera  possessed 
lytic  or  bactericidal  properties.  To  put  the  matter  to  a  test  a 
number  of  experiments,  of  the  following  nature,  were  performed. 

The  sera  of  guinea-pigs  ii  and  55  (see  Tables  i  and  2)  were  mixed 
with  constant  quantities  of  oidiomycetic  emulsion  in  dilutions  simi- 
lar to  those  used  in  method  (c)  of  the  agglutination  tests  at 
the  same  time  that  those  tests  were  made.  These  mixtures  were 
incubated  for  30  minutes,  placed  in  the  ice-chest  over  night,  and 
then  allowed  to  stand  at  room  temperature  for  three  days.  At 
the  expiration  of  that  time  the  entire  contents  of  each  tube  was 


Immunological  Reactions  of  Oidiomycosis  209 

transferred  to  an  agar  slant,  which  was  kept  in  a  horizontal  posi- 
tion for  30  minutes  in  order  to  allow  the  organisms  to  settle  on 
the  agar  surface,  and  then  placed  in  an  upright  position  in  the 
incubator  with  as  little  agitation  as  possible.  Observation  five 
days  later  (June  30,  1909)  showed  growth  on  the  agar  surface  in 
all  tubes,  both  above  and  beneath  the  surface  of  the  hquid.  The 
colonies  growing  beneath  the  surface  were  in  general  much  smaller 
than  those  above  the  fluid,  and  gave  the  impression  that  they  were 
growing  much  more  slowly.  On  July  6  the  following  notes  were 
made :  Heavy  growth  on  agar  slope  above  fluid  in  all  tubes  except- 
ing tube  containing  o .  8  c.c.  serum  (guinea-pig  55)  and  tube  contain- 
ing 0.2  c.c.  serum  (guinea-pig  11);  no  evidence  of  further  growth 
beneath  the  surface  of  the  fluid.  In  tubes  containing  o .  3  c.c, o .  6 c.c, 
0.7  c.c,  and  i.i  c.c.  serum  of  guinea-pig  55  and  that  containing 
0.5  c.c.  serum  of  guinea-pig  11,  practically  all  of  the  water  of  con- 
densation and  introduced  fluid  has  evaporated.  In  such  cases  a 
heavy  growth  extends  to  the  bottom  of  the  slope.  July  8,  no  further 
growth  beneath  the  surface  of  the  fluid  in  any  case;  otherwise  con- 
ditions are  unchanged.  Growth  is  abundant  in  all  tubes  excepting 
tubes  0.2  c.c.  and  0.5  c.c.  (guinea-pig  11)  and  tubes  0.4  c.c.  and 
0.8  c.c.  (guinea-pig  55),  in  which  the  growths  are  but  fair. 

The  sera  of  guinea-pigs  50,  51,  and  52,  together  with  the  sera 
of  three  normal  pigs,  were  next  tested  in  a  manner  similar  to  the 
above,  with  the  difference  that  in  these  cases  the  tubes  were  not 
placed  in  the  ice-chest,  that  control  tubes  of  emulsion  plus  salt 
solution  were  run  and  the  mixtures  were  incubated  for  48  hours 
before  being  transferred  to  the  agar  slope.  In  these  cases  the 
results  with  normal  and  immune  sera  were  about  the  same.  In 
all  tubes  containing  serum,  growth  was  slight  or  did  not  occur  at 
all;  while  in  the  control  salt-solution  tubes  growth  was  abundant. 

It  would  seem  from  these  experiments  that  normal  and  immune 
guinea-pig  serum  may  have  the  power  of  impairing  the  vitality 
of  oidiomycetes,  as  suggested  by  Gilkinet  for  beer  yeasts  when  sub- 
jected to  the  action  of  rabbit  serum.  However,  these  experiments 
are  regarded  as  merely  of  a  preliminary  nature,  and  it  is  hoped 
that  it  may  be  possible  to  test  the  above  conclusion  thoroughly  at 
a  later  date. 


2IO  Contributions  to  Medical  Science 

DEVIATION    OF   COMPLEMENT. 

It  cannot  as  yet  be  stated  positively  whether  or  not  normal 
or  immune  guinea-pig  serum  will  deviate  the  complement  accord- 
ing to  the  Bordet-Gengou  technic.  The  few  sera  which  have 
been  tested,  six  recently  immunized  and  one  normal,  have  given 
absolutely  negative  results.  The  experiments  have  been  carried 
on  side  by  side  with  successful  efforts  to  demonstrate  a  specific 
antibody  in  the  serum  of  guinea-pigs  immunized  to  a  bacillus  of 
the  hog  cholera  group,  so  that  in  one  way  at  least  the  work  has 
been  abundantly  controlled.  On  the  other  hand,  the  positive 
results  reported  by  other  workers  with  various  yeasts  and  molds 
make  it  still  seem  possible  that  the  technic  used  and  not  the  oidio- 
mycetes  may  be  at  fault.  One  thing  which  has  been  noticed  is 
that  O.I  c.c.  of  a  moderately  turbid  emulsion  of  oidiomycetes, 
or  O.I  c.c  of  a  freshly  made  extract,  or  0.15  c.c.  of  redissolved 
alcohohc  precipitate  of  the  extract,  is  usually  sufficient  to  inactivate 
completely  the  complement  in  o.oi  c.c.  of  guinea-pig  serum 
which  has  stood  in  the  ice-chest  over  night.  This  is,  of  course, 
but  the  expression  of  the  property,  common  to  all  yeastlike  forms, 
of  absorbing  or  destroying  complement.  With  this  fact  in  mind, 
however,  it  is  to  be  regretted  that  those  who  have  reported  posi- 
tive results  with  various  mold  fungi  have  neglected  to  give  the 
full  protocol  of  even  one  experiment.  It  might  be  of  interest  to 
compare  the  complement-binding  power  of  various  pathogenic 
and  non-pathogenic  yeast  plants;  possibly,  by  this  means,  some 
further  idea  of  the  factors  which  determine  their  power  of  resist- 
ance to  destruction  by  the  animal  body  might  be  gained. 

CUTANEOUS  AND  OPHTHALMIC  REACTIONS. 

From  the  results  of  the  skin  and  conjunctival  tubercuhn  tests  as 
applied  in  man,  it  appeared  of  interest  to  experiment  in  an  analogous 
way  with  the  oidiomycetic  extract  on  infected  guinea-pigs. 

Cutaneous  reaction. — The  method  of  applying  this  test  was  as 
follows:  A  space  on  the  abdomen  about  3  cm.  square  was  shaved 
and  washed  with  HgClj  (i :  1000)  and  alcohol  (80  per  cent).  After 
drying,  four  small  areas,  forming  the  corners  of  a  square  of  about 
I  cm.  in  breadth,  were  scarified  with  the  blade  of  a  blunt  knife 


Immunological  Reactions  of  Oidiomycosis  211 

which  had  been  dipped  into  the  following  materials:  for  area 
No.  I,  the  extract  of  organism  BIH2;  for  area  No.  2,  the  ex- 
tract of  organism  BIR;  for  area  No.  3,  0.5  per  cent  carbolic  acid. 
For  area  No.  4,  the  dry  blade  alone  was  used.  0.5  per  cent  car- 
bolic acid  was  applied  because  the  extracts  had  been  preserved 
in  that  strength  of  carbolic  acid.  In  cases  in  which  chloroform 
had  been  used  as  a  preservative,  a  0.3  per  cent  aqueous  solution  of 
chloroform  was  substituted  for  the  phenol.  In  cases  in  which 
fresh  extract  alone  was  used,  the  carbolic  acid  and  chloroform 
controls  were  omitted. 

Experiments  on  animals  suffering  from  light,  moderate,  and 
severe  infection  resulted  uniformly  and  negatively.  It  appeared 
to  make  no  difference  at  what  time  with  reference  to  the  infection 
(early,  middle,  or  late)  the  tests  were  appHed;  the  results  never 
varied.  Repeated  scarification  of  the  same  areas  after  intervals 
of  from  3  to  14  days  called  forth  no  reaction. 

Ophthalmic  reaction. — The  technic  used  in  these  experiments 
was  as  follows:  To  10  c.c.  of  the  extract  was  added  an  equal  volume 
of  absolute  alcohol.  The  resulting  precipitate  was  removed  by 
filtration,  washed  in  absolute  alcohol,  and  dried  in  a  partial  vacuum 
over  sulphuric  acid.  The  dried  material  was  redissolved  in  salt 
solution  (o. 85  per  cent  NaCl).  A  drop  of  this  solution  was  instilled 
into  the.  eye  of  the  animal  to  be  tested,  the  dose  being  repeated 
in  the  same  and  in  the  opposite  eye  after  an  interval  of  from  3  to 
10  days.  The  results  were  always  negative.  The  fresh  extract 
and  the  extract  preserved  in  phenol  and  in  chloroform  were  also 
used,  care  being  taken  to  control  with  corresponding  aqueous 
solution  of  carbolic  acid  and  chloroform,  with  similar  negative 
results. 

Apparently  then  cutaneous  and  ophthalmic  reactions  against 
the  extract  do  not  occur  in  guinea-pigs  infected  with  oidiomycosis. 

THE  FATE   OF   THE   OIDIOMYCETES  WHEN  INJECTED  INTO  THE 
PERITONEAL   CAVITY   OF   THE   GUINEA-PIG. 

Changes  in  the  peritoneal  fluids  of  recently  injected  animals. — 
The  technic  and  results  of  this  study  are  best  illustrated  by  the 
report  of  the  following  typical  experiment. 


212  Contributions  to  Medical  Science 

Guinea-pig  86  (normal)  was  given  an  intraperitoneal  injection  of  3  c.c.  of  a  rather 
dense  emulsion  in  salt  solution  (0.85  per  cent)  of  a  nine  day  agar  culture  of  oidiomyces 
at  11:10  A.M.,  April  8,  1909.  By  means  of  a  fine  glass  tube,  specimens  of  peritoneal 
fluid  were  drawn  off  at  approximately  hourly  inter\'als.  Some  of  this  material  was 
placed  upon  a  slide,  a  cover  glass  applied,  and  sealed  with  parafi&n.  The  specimen 
was  then  examined  microscopically  without  further  delay.  Cover  glass  smears  were 
made  of  other  portions  and  stained  with  the  Giemsa  mixture.  These  preparations  were 
examined  at  leisure.  The  guinea-pig  was  kept  on  his  back  for  a  few  minutes  before 
fluid  was  withdrawn,  but  was  allowed  to  run  free  in  his  cage  between  times. 

12:00  M.:  Two  specimens  of  clear,  slightly  viscid  fluid  were  obtained.  One, 
sUde  I,  was  incubated  for  30  minutes,  the  other,  slide  2,  was  examined  immediately. 

Shde  2:  Quite  a  number  of  red  cells;  a  very  small  number  of  leukocytes,  and  these 
mostl3'  mononuclear;  no  oldiomycetes  found;  red  cells  probably  due  to  trauma  inci- 
dent to  the  insertion  of  the  glass  pipette,  since  this  occasioned  a  slight  hemorrhage. 
Slide  I  is  similar  to  shde  2  in  all  respects. 

Stained  smear. — Very  few  polymorphonuclear  leukocytes,  about  in  the  same  pro- 
portion to  the  number  of  red  cells  as  in  the  blood;  the  vast  majority  of  the  leukocytes 
are  mononuclear  cells;  some  are  clearly  small  lymphocytes;  most  of  them  are  large 
mononuclear  leukocytes,  some  approaching  a  distinctly  endotheUal  type  as  judged 
by  the  abundance  of  their  cytoplasm.  Many  of  the  leukocytes  appear  to  be  disin- 
tegrating and  losing  their  staining  power.  One  organism  is  present.  It  is  free.  The 
cytoplasm  takes  an  irregular  bright  blue  stain;  the  capsule  appears  as  a  bright,  sharply 
outlined,  shiny  rim,  with  merely  the  suggestion  of  a  bluish  tinge. 

1 :  20  P.M.  Slide  3:  Still  many  red  cells;  many  times  more  leukocytes,  seemingly 
mostly  mononuclears,  a  few  organisms,  some  apparently  free,  most  of  them  surrounded 
by  from  three  to  forty  leukocj'tes;  in  some  cases  the  organism  has  been  ingested  by  a 
single  leukocyte  while  the  other  leukocj'tes  crowd  around;  in  other  cases  ingestion 
has  seemingly  not  occurred,  though  two  or  three  layers  of  leukocytes  inclose  the 
organism.     The  latter  appear  to  be  unchanged. 

Stained  smear. — Mononuclear  cells  still  predominate,  although  the  proportion  of 
polymorphonuclear  cells  has  increased.  The  leukocytes  are  frequently  clumped, 
in  some  cases  about  organisms.  The  leukocytes  in  these  clumps  seem  to  be  mostly 
mononuclears;   a  few  polynuclears  occur. 

2:20  P.M.  Slide  4:  There  are  about  the  same  number  of  red  cells  as  in  slide  3, 
but  many  more  leukocytes.  Leukocytes  occur  singly  and  in  large  and  small  groups, 
the  tendency  being  toward  the  formation  of  grape-bunch-like  masses  of  cells  so  large 
and  compact  that  it  is  impossible  to  make  out  individual  cells  excepting  at  the 
peripher>'.  Sometimes  one  can  distinguish  organisms  down  deep  in  these  masses; 
frequently  cannot  make  sure  of  the  identity  of  anything  there;  no  organisms  found 
in  very  small  masses  or  in  single  leukocytes.  In  one  instance  an  oidiomycete  can  be 
seen  within  a  single  leukocyte,  near  the  periphery  of  one  of  the  large  masses;  other 
organisms  are  present  within  that  mass,  but  one  cannot  tell  whether  they  have 
actually  been  ingested  or  not.  No  organisms  lying  free.  Exudate  is  more  viscid  than 
before,  but  does  not  seem  to  coagulate  readily.     The  oidia  are  apparently  unchanged. 

Stained  smear. — The  majority  of  the  leukoc>'tes  approach  the  transitional  type; 
there  are  quite  a  number  of  polyiiuclears;  cells  grouped  about  organisms  are  mostly 
large  mononuclear  to  transitional  in  type. 

3:20  P.M.     Slide  5:   Exudate  is  very  abundant  and  viscid;   cloudy  and  granular. 


Immunological  Reactions  of  Oidiomycosis  213 

Microscopically  very  similar  to  slide  4,  with  the  exception  of  the  greatly  increased 
number  of  leukocytes.  Many  of  the  organisms  can  be  seen  definitely  phagocyted  as 
described  in  4.  All  organisms  which  can  be  well  defined  are  found  to  be  completely 
engulfed  by  one  to  three  leukocytes,  generally  one,  the  rest  of  the  leukocytes  in  the 
mass  simply  forming  a  many  layered  cp.psule  about  the  organism  and  its  phagocytes. 
Exudate  does  not  clot  in  30  minutes. 

Stained  smear. — Large  numbers  of  polynuclear  cells,  otherwise  similar  to  4.  Cyto- 
plasm of  cells  grouped  about  oidiomycetes  is  fused  into  a  single  mass  in  some  cases; 
in  others  the  cells  are  simply  connected  by  strands  of  cytoplasm,  giving  a  vacuolated 
appearance  to  the  region  between  the  cells. 

4:20  P.M.  Slide  6:  Exudate  is  abundant.  It  is  still  richer  in  leukocytes  than 
before;  there  are  quite  a  number  of  phagocyted  organisms.  The  leukocytes  do  not 
seem  to  be  bunched  so  strikingly  as  in  previous  slides;  the  oldia  are  inclosed  within 
from  I  to  10  or  a  dozen  leukocytes;  the  leukoc>'tes  are  more  compact  about  the 
organisms  than  formerly,  and  instead  of  appearing  like  a  bunch  of  grapes,  that  is, 
grouped  rather  loosely  about  phagocyted  organisms,  the  leukocytic  mass  now  has 
the  appearance  of  segmenting  frog  eggs,  all  of  the  cells  fitting  closely  to  each  other. 
None  of  the  very  large  bunches  of  leukocytes  are  seen  which  were  so  striking  in  slides 
3,  4,  and  5.     Exudate  does  not  coagulate  readily. 

Stained  smear. — Polynuclears  predominate  largely,  and  seem  to  be  the  phago- 
cytes. 

5:20  P.M.  Slide  7:  Large  clumps  of  leukocytes  about  oidiomycetes  do  not 
appear.  Frequently  a  single  organism  may  be  found  within  one  leukocyte;  almost 
never  are  more  than  five  or  six  leukocytes  concerned,  excepting  where  a  number  of 
organisms  occur  together,  in  which  case  the  whole  mass  may  be  fairly  large,  though  the 
number  of  leukocytes  per  organism  is  apparently  not  what  it  was  in  earlier  slides. 
The  organisms  appear  to  be  unchanged. 

Stained  smear. — Similar  to  6.  The  nuclei  of  the  leukocytes  appear  to  be  under- 
going a  process  of  karyorrhexis. 

6: 20  P.M.  Slide  8:  Similar  to  the  above  in  the  main.  One  important  difference, 
i.e.,  leukocytes  immediately  surrounding  organisms  seem  to  be  breaking  up;  they 
are  losing  their  distinctness  of  outline  and  the  granules  are  spreading  out  diffusely 
into  the  surrounding  zones.  Oidia  are  all  budding,  this  condition  seeming  to  be  a  little 
more  marked  than  in  earlier  specimens  of  the  exudate. 

Stained  smear. — Similar  to  7,  more  leukocytic  changes.  It  is  hard  to  determine 
whether  the  phagocytic  cells  are  mononuclear  or  polymorphonuclear;  it  seems  best  to 
regard  them  as  mononuclear,  although  doubtful. 

March  9,  1909.  9:30  a.m.  SHde  9:  Leukocytes  grouped  about  organisms 
seem  to  be  fragmenting.  Leukocytes  are  scattered  evenly  over  the  field.  Aggre- 
gations of  20-30  about  an  organism,  especially  a  budding  form,  are  still  to  be  found; 
those  leukocytes  in  the  center  of  such  masses  seem  to  be  more  or  less  fused  with 
each  other.  Oidiomycetes  contain  variable  numbers,  generally  inconsiderable  (10-20), 
of  small,  somewhat  highly  refractive,  spherical  bodies,  arranged  as  a  rule  near  the 
periphery  of  the  cell  within  the  capsule.  Frequently  they  are  scattered  irregularly 
through  the  cell.  Many  of  the  leukocytes  in  diverse  parts  of  the  field  contain  very 
similar  granules.     Sometimes  organisms  appear  to  be  full  of  such  bodies. 

Stained  smear. — In  a  number  of  instances  it  can  be  seen  that  the  phago- 
cytosis is  undoubtedly  by  large  mononuclear  cells.     One  group  is  especially  noticeable; 


214  Contributions  to  Medical  Science 

it  consists  of  a  budding  organism  inclosed  by  four  leukocytes.  The  leukocytes  are 
arranged  radially  about  the  organism.  The  peripheral  three-quarters  of  the  cyto- 
plasm of  each  cell  is  independent  and  distinct,  the  inner  fourth,  however,  has  merged 
with  a  corresponding  part  of  the  other  three  to  surround  the  oldium  by  a  uniform 
cytoplasmic  mass.  The  disposition  of  the  cells  reminds  one  a  little  of  the  structure 
of  a  four-leaf  clover.  The  nuclei  of  the  leukocytes  stain  purple;  the  cytoplasm, 
which  is  moderately  vacuolated,  a  robin's-egg  blue.  The  intracapsular  portion  of  the 
organisms  takes  an  irregularly  distributed,  deep  to  light  blue  stain;  the  capsule  is 
clear,  homogeneous,  and  of  a  very  light  blue  color. 

10:30  A.M.  Slide  10:  Cells,  unstained,  appear  like  large  mononuclears  for  the 
most  part.  Groups  of  leukocytes  are  sometimes  found  arranged  radially  about  an 
indistinct,  somewhat  homogeneous  central  mass  in  which  it  seems  one  can  make  out 
leukocytic  shadows.  The  cells  at  the  periphery  of  such  masses  are  more  or  less  cone- 
shaped,  apex  inward,  nucleus  at  the  base,  peripheralward.  The  apex  is  drawn  out 
into  a  narrow  projection  which  loses  itself  in  the  central  area.  Budding  oldia  are 
found  packed  full  of  granules;  then  again  non-budding  forms  occur  which  contain  a 
very  indistinct,  faintly  granular,  slightly  refractive  material.  Organisms  are  still 
engulfed  by  leukocytes,  though  it  looks  sometimes,  especially  in  the  case  of  the  bud- 
ding, strongly  granular  forms,  as  though  the  leukocytes  were  beginning  to  break  away 
from  the  organism  and  its  immediate  phagocyte.  Again  we  find  budding,  markedly 
granular  organisms  in  the  center  of  such  leukocj'tic  masses  as  are  described  above. 
Granules  may  or  may  not  be  present  in  the  buds  of  such  organisms.  Phagocyted 
oldia  are  observed  in  which  the  cytoplasm  seems  to  have  become  homogeneous  and 
is  divided  into  two  or  three  segments. 

Stained  smear. — Sometimes  the  nucleus  and  cytoplasm  of  the  leukoc>'tes  are 
markedly  vacuolated,  and  the  cell  appears  to  be  disintegrating.  In  a  few  instances 
the  intracapsular  portion  of  the  oidia  stains  much  less  deeply  than  usual  while  the 
immediate  leukocytes  are  quite  well  preserved.  Polymorphonuclear  cells  are  much 
more  numerous  than  the  other  varieties  of  leukocj'tes,  but  appear  to  take  no  part  in 
the  phagocytosis.  Tjpical  small  Ij-mphocytes  are  very  few  in  number.  The  large 
mononuclear  cells  with  a  great  deal  of  cytoplasm  form  the  great  bulk  of  mononuclear 
leukocytes. 

2:30  P.M.  Slide  11:  Essentially  similar  to  10.  One  organism  is  present  which 
has  an  unusually  thick  capsule  (2-3  times  ordinary  thickness).  The  outside  of  the 
capsule  is  smooth,  the  inside  irregular.  This  organism  has  one  small  bud.  Other 
oidiomycetes  in  the  same  mass  appear  normal. 

Stained  smear. — Phagocytosis  is  not  exclusively  by  mononuclear  leukocytes;  a 
few  polymorphs  may  be  included  in  the  plasmodial  masses. 

5:00  P.M.  SHde  12:  Leukocytic  groups  occur  about  organisms  as  in  the  two 
preceding  slides.  Fusion  of  leukocytes  and  extrusion  of  granules — breaking  up  of 
leukocytes  ? — is,  if  anything,  more  marked.  As  many  as  seven  budding  organisms  are 
found  in  one  leukocytic  mass.  Oidia  seem  to  be  budding  and  multiplying  since  we 
now  find  two,  three,  or  four  cells  grouped  together,  sometimes  in  chains — mother  cell, 
daughter  cell  ? — sometimes  in  pairs  nearly  always  giving  evidence  of  budding.  One 
organism  is  found  with  two  buds.  Also  find  free  organisms  for  the  first  time  since  the 
second  hour.  One  of  these  is  rather  thick-walled  and  is  packed  with  25-30  spherical, 
homogeneous  bodies.  The  spherules  are  not  so  numerous  but  that  each  can  be  dis- 
tinguished. 


Immunological  Reactions  of  Oidiomycosis  215 

Stained  smear. — Similar  to  ii.     Phagocytic  cells  seem  to  be  breaking  up. 

April  9,  1909.  8:20  A.M.  Slide  13:  Many  free  organisms.  Also  many  large 
accumulations  of  leukocytes  in  the  midst  of  which  groups  of  organisms,  two  to  six  in 
a  group,  occur.     Exudate  is  relatively  scanty. 

Stained  smear. — Similar  to  12.  Mononuclear  cells  seem  to  be  relatively  more 
numerous  than  in  last  few  specimens. 

April  12,  1909.  11:30  A.M.  Slide  14:  (Pig  seems  somewhat  asthenic.  Tissues 
are  swollen  at  abdominal  wound.  Wound  is  closed  but  bleeds  readily.  Small,  pin- 
head  nodules  are  palpable  in  right  testicle.  Left  testicle  is  hardened;  nodules 
similar  to  those  in  the  right  testicle  are  found.)  Exudate  is  very  scanty.  One  small 
drop  obtained  which  shows  very  marked  concentration  of  leukocytes  about  organ- 
isms, giving  an  appearance  very  similar  to  typical  giant  cells  of  the  foreign  body 
type. 

Stained  smear. — Mononuclear  cells  only,  big,  endothelial-like  cells  for  the  most 
part.  They  occur  in  huge  masses,  surrounding  numerous  organisms.  All  leukocytes 
in  these  masses  seem  to  be  losing  their  staining  power;  cytoplasm  and  nucleus  is 
vacuolated. 

This  specimen  was  the  last  that  could  be  obtained.  Slide  14  was  allowed  to  stand 
at  room  temperature.  Examined  the  next  day,  it  was  found  that  long  hyphae  had 
grown  out  from  many  of  the  organisms  which  were  inclosed  in  giant  cells. 

The  guinea-pig  was  etherized  two  days  after  the  making  of 
sHde  14.  At  autopsy  the  peritoneum,  visceral  and  parietal,  was 
dotted  with  grayish  nodules  from  the  size  of  minute  points  to 
nodules  having  a  diameter  of  from  5  to  7  mm.  The  omentum  was 
practically  a  mass  of  such  nodules.  The  testicular  surfaces  also 
presented  numerous  grayish  nodules,  the  largest  being  about  i 
mm.  in  breadth.  There  was  no  excess  of  fluid  in  the  peritoneal 
cavity.  The  nodules  at  first  sight  seemed  to  be  retroperitoneal. 
Attempts  to  remove  them  showed  that  most  could  be  peeled  off 
without  a  great  deal  of  difficulty,  leaving  a  roughened  opaque 
surface,  while  a  few  were  considerably  more  adherent  and,  when 
removed,  left  a  roughened  opaque  surface  on  which  could  be  seen 
an  occasional  bleeding  point.  On  cutting  the  nodules,  some,  es- 
pecially the  larger  ones,  were  found  to  have  soft  centers;  smears 
of  this  material  showed  polymorphonuclear  leukocytes  and  oidiomy- 
cetes.  Microscopically  the  nodules  fixed  in  Zenker's  fluid,  imbedded 
in  celloidin  and  stained  with  hematoxylin  and  eosin,  were  found  to 
consist  of  masses  of  fibrin  and  leukocytes.  The  leukocytes  were 
mostly  of  the  large  mononuclear,  endothelial-like  type,  although  in 
some  isolated  spots  polymorphonuclear  leukocytes  predominated. 
Oidiomycetes  were  present  in  large  numbers,  usually  in  the  center 


2i6  Contributions  to  Medical  Science 

of  the  nodule,  and  inclosed  usually  within  cells  having  from  i  to  20 
oval,  well  stained,  peripherally  placed  nuclei.  As  a  rule  the  nuclei 
numbered  from  3  to  8. 

The  abundance  of  the  cytoplasm  of  such  cells  was  in  direct 
proportion  to  the  number  of  nuclei  and  took  a  marked  uniform 
eosin  stain  with  suggestion  of  a  very  finely  granular  structure. 
Sometimes  the  organisms  were  apparently  free  in  the  nodule;  in 
such  cases  they  formed  the  center  of  a  small,  circular,  sharply 
circumscribed  accumulation  of  polymorphonuclear  leukocytes. 
There  were  no  evidences  of  necrosis.  The  oidiomycetes  them- 
selves presented  a  variety  of  appearances.  The  budding  forms 
frequently  occupied  the  center  of  the  small  abscesses  just  men- 
tioned; they  presented  a  brightly  shining,  unstained,  double- 
contoured  external  membrane,  within  which  was  an  irregularly 
distributed,  blue-stained,  somewhat  granular  material  which 
seemed,  however,  to  form  a  lining  layer  just  within  the  limiting 
membrane  and  not  to  be  diffused  to  any  great  extent  throughout 
the  cell.  In  other  organisms,  the  double-contoured  membrane 
was  unchanged  and  the  material  within  the  cell  still  took  a  blue 
stain;  the  latter,  however,  had  seemingly  shrunk  away  from  the 
outer  wall  at  all  but  one  point,  leaving  a  clear,  unstained,  new-moon 
shaped  space.  In  still  other  organisms,  this  material  had  seem- 
ingly shrunk  still  more  and  was  beginning  to  take  an  eosin  stain; 
in  others,  it  was  beginning  to  lose  its  granular  structure  and  to 
assume  a  homogeneous  appearance,  taking  a  sharp  eosin  stain. 
In  such  organisms  as  this,  the  central  portion  had  sometimes 
become  completely  separated  from  the  cell  membrane  so  that  the 
new-moon  shaped  space  had  grown  into  a  complete  circle;  in  other 
organisms,  an  equally  pink-stained,  homogeneous  central  portion 
was  still  in  contact  with  the  external  membrane  at  one  point,  lead- 
ing one  to  imagine  that  possibly  the  complete  separation  just 
described  might  be  more  apparent  than  real  owing  to  the  plane  in 
which  the  organism  had  been  cut. 

Quite  frequently  no  sign  of  "shrinking"  was  observed,  the 
cells  being  a  homogeneous  pink  without  other  modification.  In 
those  organisms  which  took  the  blue  stain,  it  appeared  as  though 
the  unstained  portions  within  the  capsule  represented  the  homo- 


Immunological  Reactions  of  Oidiomycosis  217 

geneous  spherules  described  in  the  fresh  specimens  while  the 
stained  parts  represented  what  might  be  termed  the  interspheru- 
lar  substance.  A  somewhat  similar  series  of  changes  occurring 
in  yeast  intra  vitam  has  been  described  by  Potron.^^ 

The  peritoneum  extended  intact  beneath  most  of  the  nodules. 
The  cells  of  the  peritoneum,  however,  had  assumed  a  marked 
cuboidal  or  rounded  shape  and  seemed  to  have  broken  contact 
with  each  other.  In  a  few  instances  a  typical  organization  was 
proceeding  from  the  sub-peritoneal  tissues  into  the  overlying 
oidiomycetes  containing  exudate. 

The  kidneys  were  normal,  as  were  the  other  organs.  It  will 
be  remembered  that  some  of  the  organisms  in  the  peritoneal 
exudate  of  shde  14  grew.  Oidiomycetes  developed  in  pure  cul- 
ture from  material  taken  from  nodules  at  autopsy. 

It  does  not  appear  to  be  necessary  to  report  further  experi- 
ments in  detail.  We  shall  summarize  the  course  of  events  in  the 
peritoneal  cavity  of  a  guinea-pig  following  the  injection  of  an 
emulsion  of  living  oidiomycetes  in  0.85  per  cent  NaCl  solution, 
basing  our  summary  on  numerous  experiments  such  as  the  fore- 
going, on  the  autopsy  findings  of  pigs  dying  or  killed  during  the 
course  of  an  experimental  infection,  and  on  observations  made  at 
exploratory  operations,  as  follows:  There  is  first  a  great  transuda- 
tion of  fluid  poor  in  fibrinogen  and  leukocytes  into  the  peritoneal 
cavity.  The  few  leukocytes  present  are  mostly  large  mononuclears, 
though  lymphocytes  and  polymorphonuclear  leukocytes  occur. 
After  one  or  two  hours  the  leukocytes  begin  to  accumulate 
rapidly,  and  at  the  end  of  three  or  four  hours,  the  peritoneal 
fluid,  at  first  thin  and  watery,  becomes  very  cloudy  and  quite 
viscid.  This  cloudiness  and  viscidity  gradually  increase  as  the 
fluid  becomes  more  and  more  scanty,  until  finally,  after  three  or 
four  days,  no  more  fluid  can  be  obtained.  If  one  opens  the  abdom- 
inal cavity  of  a  guinea-pig  24  hours  after  the  injection  of  a  large 
dose  of  oidiomycetes,  he  finds  an  acute,  diffuse,  suppurative,  and 
fibrinous  peritonitis.  The  peritoneum  is  covered  with  a  thick 
layer  of  turbid,  sticky  fluid  in  which  are  occasional  yellowish-gray 
clumps.  These  clumps  are  soft  and  may  be  easily  lifted  from  the 
peritoneal  surface;  in  fact,  they  can  hardly  be  said  to  be  adherent. 


2i8  Contributions  to  Medical  Science 

The  microscopical  appearance  of  this  exudate  has  been  described 
above.  The  clumps  consist  of  accumulations  of  phagocytic  cells 
and  groups  of  cells — the  "rosettes"  of  Skchi wan— supported 
by  a  scanty  fibrinous  mesh  work.  After  four  days  very  little 
fluid  will  be  found ;  the  peritoneum  may  have  a  dull  grayish  tinge 
or  may  be  about  normal  in  appearance;  it  surely,  however,  will 
present  numerous  raised,  convex,  grayish  nodules  0.5  to  5  mm. 
in  diameter  and  sometimes  whitish  patches  as  much  as  i  cm.  in 
greatest  extent,  but  these  latter  are  not  usual.  As  a  rule,  nodules 
and  patches  are  easily  detachable,  leaving  a  dull,  rough  surface 
which  sometimes  may  be  slightly  granular.  They  consist  of 
masses  of  single  large  mononuclear  leukocytes,  some  containing 
organisms,  of  polymorphonuclear  leukocytes  and  masses  of  the 
phagocytic  "rosettes,"  in  a  fibrinous  meshwork.  As  the  obser- 
vations are  extended  to  include  lesions  at  longer  and  longer 
intervals  following  the  injection,  it  is  found  that  the  nodules, 
beginning  about  the  fifth  day,  become  more  and  more  difficult  to 
remove,  and,  when  removed,  become  more  and  more  prone  to 
leave  a  bleeding  surface.  Some  seem  to  sink  down  into  the  tissue 
upon  which  they  at  first  rested;  the  peritoneum  grows  over  them; 
they  become  less  and  less  prominent,  and  finally  disappear  entirely 
in  the  course  of  15  to  30  days.  Other  nodules — and  this  seems  to 
be  especially  true  of  nodules  in  the  testicles  following  the  intra- 
peritoneal injection  of  o.i  gm.  of  organism  or  more — commonly 
grow  progressively  larger  for  from  4  to  10  days,  become  somewhat 
soft  and  doughy,  and  then,  in  favorable  cases,  gradually  harden,  de- 
crease in  size  and  disappear  entirely  within  40  days.  In  unfavorable 
cases  the  process  of  enlargement  and  softening  continues  until,  as  in 
guinea-pig  75  (see  p.  205),  the  scrotum  becomes  greatly  distended, 
the  overlying  skin  becomes  involved,  and  the  lesion  finds  an  external 
opening.  Microscopically,  in  the  first  type,  blood-vessels  are  ob- 
served to  grow  into  the  "rosette"  masses  from  underlying  tissues, 
connective  tissue  is  laid  down,  the  nodules  become  young  connective- 
tissue  growths,  and  presently,  the  "rosettes"  formed  from  free, 
phagocytic  macrophages  are  transformed  into  morphologically 
typical  Langhans  giant  cells  of  inflammatory  granulation  tissue 


Immunological  Reactions  of  Oidiomycosis  219 

merely  by  this  replacement  of  their  supporting  fibrinous  reticulum 
by  a  meshwork  of  capillaries  and  their  accompanying  connective- 
tissue  cells  (see  Fig.  5). 

In  the  case  of  very  large  nodules  such  as  those  which  formed 
in  the  peritoneal  cavities  of  guinea-pigs  13,  37,  38,  and  56  (see  p. 
205)  the  connective-tissue  growth  may  be  so  extensive  as  to  cut 
off  its  own  blood  supply,  thick-walled  cysts  being  formed  which 
contain  a  pus-Hke  fluid.  This  material  is  not  pus,  however,  but 
consists  of  cellular  detritus,  and,  in  the  earlier  stage,  contains 
oidiomycetes  showing  various  degrees  of  degeneration.  In  stained 
sections  of  this  necrotic  material  from  guinea-pig  13,  autopsied 
100  days  after  the  last  injection,  one  finds  oidiomycetes  which 
are  practically  normal  in  appearance,  others  have  assumed  appear- 
ances similar  to  those  described  in  the  nodules  of  guinea-pig  86, 
while  others  have  gone  still  farther  in  that  the  cell  membrane  has 
either  become  thickened,  homogeneous,  eosin-stained,  irregular  in 
outline,  or  in  other  instances  has  disappeared  leaving  merely  the 
inner  homogeneous,  eosin-stained  spheres.  Microscopical  exami- 
nation of  the  unstained  detritus  reveals  bodies  very  similar  to 
organisms  in  appearance  but  which  are  soluble  in  ether.  In 
specimens  from  other  nodules  of  similar  age,  guinea-pig  56,  for 
instance,  autopsied  116  days  after  last  injection,  the  ether-soluble 
bodies  are  the  only  things  which  occur  that  in  any  way  resemble 
oidiomycetes.  Whether  or  not  they  bear  any  direct  relation  to 
the  organisms  has  not  been  determined. 

In  the  case  of  those  nodules  which  become  progressively  larger 
and  tend  to  soften,  the  bulk  of  the  leukocytes  consists  of  poly- 
morphonuclears. In  such  masses,  organisms  commonly  occur 
free;  frequently  they  remain  inclosed  in  Langhans  giant  cells 
which  thus  appear  in  stained  sections  as  eosin-stained  islets  in  the 
mass  of  blue-lobed  nuclei.  Such  nodules  occur  to  some  extent 
in  the  early  stages  of  all  cases,  but  in  the  later  stages  of  fatal 
cases  only. 

Mention  has  been  made  of  nodules  "in  the  testicles."  This 
statement  is  not  strictly  correct  since  most  nodules  palpated  ''in 
the  testicles"  are  merely  in  the  peritoneal  and  fibrous  tunics. 


220  Contributions  to  Medical  Science 

Even  in  the  most  advanced  cases  the  testicle  itself  may  be  and 
generally  is  unharmed,  as  in  guinea-pig  75  (see  p.  205)  in  which 
at  autopsy  the  testicles  were  found  normal. 

Besides  the  peritoneal  lesions,  macroscopic  nodules  have  been 
found  in  the  liver  and  in  the  lungs.  In  the  liver  they  appear 
as  white  to  grayish-red  patches  1-5  mm.  in  cross-sectiorf,  rather 
sharply  demarkated.  In  the  lungs  they  appear  as  translucent 
droplets  0.5-2  mm.  in  diameter,  occurring  both  sub-pleurally  and 
on  the  cut  surface.  The  animals  seem  to  have  died  before  larger 
nodules  had  time  to  develop. 

Under  the  microscope  in  sections  stained  with  hematoxylin 
and  eosin,  tj^pical  liver  nodules  appear  as  follows: 

Section  of  the  liver  of  guinea-pig  53  (for  history,  see  p.  195;  see  also  Plate  i). 
Slight  passive  congestion;  sUght  central  fatty  infiltration;  there  is  one  area  with  a 
diameter  of  4-6  liver  lobules  in  which  the  parenchjTna  cells  seem  to  have  been  replaced 
by  widely  dilated,  erythrocyte-filled  sinuses,  and  oidiomjxetes  inclosed  in  cells  having 
from  I  to  20  nuclei.  The  multinucleated  cells  resemble  closely  foreign-body  giant 
cells,  excepting  that  the  nuclei  tend  to  be  more  rounded  than  those  commonly  seen 
in  the  latter  cells,  and,  when  oval  in  shape,  their  long  axis  tends  to  run  parallel  with 
the  surface  of  the  cell  rather  than  centralward.  This  area  is  sharply  marked  off  from 
the  hver  tissue  by  a  narrow  layer  of  parallel,  wa\y,  pink-stained  fibers,  peripheral  to 
which  is  quite  a  marked  infiltration  of  mononuclear  cells,  mostly  of  the  hmphoid 
type,  though  many  are  cells  with  abundant  cytoplasm.  The  neighboring  parench>Tna 
cells  are  normal  in  most  cases,  although  occasional  islets  of  liver  cells  in  which  are 
found  all  stages  of  degeneration,  even  to  that  of  complete  necrosis,  appear  in  the 
border  tissue. 

In  the  lungs,  the  nodules  occur  principally  in  the  neighborhood 
of  the  bronchi  or  larger  blood-vessels.  They  consist  of  dense 
accumulations  of  round  and  epitheHoid  cells  with  an  organism 
at  rare  intervals;  no  necrosis;  no  edema;  no  exudation  into 
alveoli.     Aside  from  the  nodules  the  lungs  are  normal. 

phagocytosis  in  vitro. 

The  leukocytes. — Guinea-pigs  were  given  intraperitoneal  injec- 
tions of  a  thick  suspension  of  sterile  aleuronat  in  0.85  per  cent 
NaCl  solution.  From  examination  of  the  exudate  at  hourly 
intervals  it  was  found  that,  after  a  lapse  of  from  12  to  15  hours, 
the  exudate  was  very  rich  in  leukocytes  and  was  still  sufficient  in 
quantity  to  be  easily  obtained.     Of  the  leukocytes,  from  25-50 


Immunological  Reactions  of  Oidiomycosis  221 

per  cent  were  large  mononuclears,  the  remainder  being  mostly 
polymorphonuclear  neutrophiles  and  eosinophiles.  Both  the 
resh  exudate  and  ''washed"  leukocytes  were  used.  The  "washed" 
leukocytes  were  obtained  by  washing  the  fresh  exudate  first  in 
sodium  citrate  solution  and  then  in  one  or  two  changes  of  physio- 
logical salt  solution  according  to  the  ordinary  opsonic  technic. 
The  suspension  was  then  made  up  to  the  original  volume  of  the 
exudate  by  the  addition  of  0.85  per  cent  NaCl  solution.  The 
fresh  exudate  clotted  readily,  so  that,  in  using  it,  rapid  work  was 
necessary. 

Exudate  serum. — This  was  obtained  by  allowing  the  fresh 
exudate  to  clot  spontaneously  in  test-tubes,  centrifuging  imme- 
diately, and  removing  the  serum  by  means  of  a  glass  pipette. 

The  o'idiomycetes  were  placed  in  a  bottle  of  normal  salt  solution 
and  shaken  vigorously  in  a  shaking  machine  for  several  hours 
before  being  used,  in  order  to  break  up  any  clumps  which  might 
be  present.  An  emulsion  of  the  organisms  in  salt  solution  was  then 
made  which  contained  approximately  5,000  organisms  per  c.mm. 
In  order  to  do  this,  counts  were  made  of  the  organisms  in  the 
emulsion  in  the  shaking  bottle  by  means  of  the  Thoma-Zeiss  hemo- 
cytometer,  and  this  emulsion  was  then  diluted  to  the  volume 
required  as  shown  by  the  excess  of  organisms  over  the  5,000  per 
c.mm.  which  was  desired. 

Method. — The  observations  were  made  on  hanging-drop  prep- 
arations. The  various  materials  were  mixed  on  clean  cover  glasses 
in  tripHcate,  in  the  dilutions  called  for  by  the  protocol,  mounted 
on  hollow  ground  slides,  and  the  cover-slip  sealed  to  the  sUdes 
with  paraffin.  The  preparations  were  incubated  at  37°  C.  and 
observed  after  30  minutes,  after  3  hours,  and  after  24  hours.  No 
stain  was  employed. 

The  first  attempt  was  directed  to  determining  (i)  whether  or 
not  phagocytosis  would  occur  in  vitro,  using  {a)  the  fresh  exudate 
and  {b)  washed  leukocytes,  and  (2)  the  possible  influence  of  guinea- 
pig  serum  upon  the  process. 

Several  preliminary  experiments  of  which  the  following  is  a 
typical  example  were  performed : 


222 


Contributions  to  Medical  Science 


TABLE  6. 

EXPERI»£ENX  IN  PHAGOCYTOSIS. 


SUde  No. 

Organisms 
(Units  of 
Emulsion) 

Fresh 
Exudate 
(Units) 

Washed  Leu- 
kocytes 
(Units) 

Normal  Blood 
Serum  (Units) 

Exudate 
Serum  (Units) 

NaCl  (o.8s 

per  cent) 

(Units) 

o 

I 

2 

3 

4 

s 

6 

7 

8 

9 

10 

I 

I 
o 
I 
o 
I 
o 
I 
I 
I 
I 

o 
o 
I 
o 
I 
o 
I 
o 
o 
o 
o 

20 
lO 

0 

8 
o 
6 

I 
4 

2 

o 
o 

o 
o 
o 
o 
I 
o 

0 

o 
o 
o 
o 

o 
o 

I 

2 
O 
4 
O 

6 
8 

lO 
0 

The  final  examination  of  these  slides  after  24  hours  at  37°  C. 
disclosed  the  following  conditions: 

Slide  o:  Well  rtuxed;  many  red  cells;  leukocytes  occur  singly  but  also  very 
frequently  in  groups  of  two  to  a  dozen  or  more — most  commonly  three  or  four  cells  per 
group.  Organisms  may  or  may  not,  infrequently  not,  be  found  within  these  groups. 
The  rtile  appears  to  be  for  the  leukocytes  to  pay  no  attention  to  the  oidiomycetes. 
Very  rarely  a  single  leukocyte  may  be  seen  which  has  partially  or  totallj'  engulfed  an 
organism. 

Shde  I :  Organisms  oc- 
curring singlj^  are  seen  only 
within  leukocytes.  Organ- 
isms occurring  in  clumps  are 
always  surrounded  and,  to 
be  perceived  by  careful 
focusing,  engulfed  by  leuko- 
cytes. Leukocytic  clumps 
are  found  in  the  absence  of 
organisms,  but  not  to  the 
extent    observed    in    slides 

2,  4,  and  6  (see  below).  Many  large  leukocytes  with  abundant  cytoplasm  occur;  they 
are  the  actively  phagocytic  cells.  There  are  many  red  blood  corpuscles,  but  ver>-  few 
of  them  have  been  ingested. 

Slide  2:  Leukocytes  and  organisms  are  quite  numerous.  Both  types  of  cells, 
leukocytes  and  oidiomycetes,  occur  singly,  by  twos  and  threes,  and  in  large  groups, 
the  organisms  to  a  greater  extent  than  the  leukocytes.  The  cells  are  well  intermixed 
and  frequently  approximate  each  other,  but  this  is  not  marked  enough  to  suggest 
phagocytosis  or  even  positive  chemiotaxis. 

Slide  3:  Very  similar  to  slide  i,  excepting  that  there  is  more  extensive  ingestion 
of  red  cells.  In  some  cases  leukocytes  are  merely  grouped  about  organisms,  in  others 
there  is  not  only  grouping,  but  phagocytosis. 

Slide  4:  Similar  to  slide  2,  excepting  that  two  leukocj^tes  are  present  which 
appear  to  be  phagocytic.     Each  has  partly  surrounded  an  elongated  blastomycete. 

Slide  5 :  Many  red  blood  cells  are  present.  A  good  many  of  them  have  been 
phagocj'ted.     Organisms  are  present  in  fair  numbers  and  occur  for  the  most  part 


Fig.  I. — Types  of  phagocytic  cells:  a,  erythrocytes;   6,  organ- 
isms; c,  cell  nucleus. 


Immunological  Reactions  of  Oidiomycosis 


223 


within  single,  or  plasmodial  masses  of,  leukocytes.     It  is  noticeable  that  the  leuko- 
cytes which  take  up  the  red  blood  corpuscles  also  take  up  the  organisms.    It  is  common 

to  find  a  leukocyte  which  contains  2,  3, 
or  even  4  red  cells  in  addition  to  an  or- 
ganism. Such  large  cells  seem  to  be  less 
numerous  here  than  in  shde  3.  They 
assume  bizarre  shapes — greatly  elongated, 
spindle  forms,  star-forms,  and  various 
other  irregular  outlines,  with  long,  slender 
projections.  This  is  especially  noticeable 
about  the  edges  where  the  drop  is  thin. 

Shde  6 :  Same  general  appearance  as 
slide  4. 

Shde  7:    Fewer  cells  of  all  kinds; 
otherwise  similar  to  slide  5. 

Slide  8:  Cells  of  all  kinds  seem  to  be 
still  fewer  than  in  slide  7.  Absolutely 
free  organisms  are  quire  rare;  on  the 
other  hand,  ingestion  is  not  always  com- 
plete and  many  of  the  groupings  suggest 
that  shown  in  Fig.  2.  This  appears  to  be  due  in  some  degree  to  a  dearth  of  the  big 
leukocytes,  since  where  they  occur  ingestion  is  as  complete,  as  a  rule  (there  are  ex- 
ceptions), as  their  bulk  makes 
possible,  as  is  suggested  in  the 
accompanying  sketch. 

Red  blood  corpuscles  have 
been  phagocyted  quite  freely 
but  by  no  means  to  the  degree, 
relatively,  that  is  evident  in 
the  case  of  the  oldiomycetes. 

Slide  9 :  Complete  ingestion 
of  single  and  budding  organisms  occurs  with  fair  frequency;  on  the  other  hand,  in 


Fig.  2. — Sketches  of  two  strikingly  similar 
oidiomycetic  groups  to  illustrate  the  difference 
between  (a)  mere  grouping  of  leukocytes,  and  (6) 
grouping  with  phagocytosis;  (c)  shows  a  leukocyte 
containing  two  organisms.  These  sketches  also 
illustrate  the  type  of  ceU  which  is  actively 
phagocytic. 


Fig.  6. 


many  instances  the  leukocytes  seem  to  be  entirely  indifferent  to  the  oldia.     In  plas- 
modial masses  which  are  in  relation  to  organisms  there  is  generally  more  or  less  com- 


224  Contributions  to  Medical  Science 

plete  phagocytosis;  contrarywise,  places  are  not  hard  to  find  where  mere  grouping, 
with  little  or  no  phagocytosis,  occurs. 

Slide  id:  Conditions  here  are  very  similar  to  those  in  shde  o.  If  anything, 
phagocytosis  may  be  slightly  less  marked,  something  of  a  hair-spitting  distinction, 
however. 

From  this  experiment  it  appeared  (i)  that  the  leukocytes  of 
the  fresh  exudate,  either  without  the  addition  of  normal  serum  or 
when  mixed  with  excessive  quantities  of  normal  serum,  do  not 
phagocyte  oidiomycetes  as  well  as  such  leukocytes  plus  moderate 
quantities  of  serum;  (2)  that  washed  leukocytes  do  not  phago- 
cyte oidiomycetes  readily,  when  suspended  in  simple  salt  solu- 
tion, when  suspended  in  salt  solution  plus  normal  serum,  or  salt 
solution  plus  exudate  serum;  (3)  that  phagocytosis  is  carried  on 
in  the  fresh  exudate  in  vitro  by  cells  of  the  same  type  as  those  which 
are  responsible  for  the  major  part  of  the  work  in  the  peritoneal 
cavity. 

An  extensive  series  of  further  experiments  was  planned  with 
the  idea  of  testing  thoroughly  the  above  deductions.  Unfortu- 
nately, the  adoption  of  the  mycelial  mode  of  growth  by  the  organ- 
ism interrupted  the  work  while  it  was  still  far  from  being  complete, 
so  that  the  results  reported  below  must  be  regarded  as  purely 
tentative. 

The  summary  of  events  follows:  (i)  It  appears  that  the  leuko- 
cytes of  the  undiluted  fresh  exudate  are  very  actively  phagocytic; 
complete  phagocytosis  is  the  rule  within  30  minutes  at  37°  C; 
that  is,  as  a  rule,  every  organism  in  the  preparation  will  have 
been  ingested  within  that  space  of  time.  The  leukocytes  com- 
monly form  a  plasmodial  mass  about  the  oidiomycetes,  the  mass 
being  surrounded  by  a  narrow  clear  zone  of  fairly  uniform  width 
which  separates  it  from  the  surrounding  leukocytes.  If  the 
exudate  is  diluted  by  salt  solution,  normal  serum,  immune  serum, 
or  exudate  serum,  the  degree  of  phagocytosis  seems  to  decline, 
roughly,  with  the  increase  of  the  dilution.  In  other  words,  the 
more  widely  the  organisms  are  separated  from  the  leukocytes,  the 
less  apt  is  phagocytosis  to  occur.  The  organisms  do  not  seem  to  be 
strongly  chemiotactic  in  the  sense  that  leukocytes  will  be  attracted 
to  them  from  a  distance,  the  ingestion  of  the  oidiomycetes  by  the 
leukocytes  seems  to  be  a  sort  of  contact  phagocytosis.     The  sera 


Immunological  Reactions  of  Oidiomycosis  225 

do  not  encourage  phagocytosis  to  any  greater  extent  than  does 
physiological  salt  solution. 

(2)  As  was  indicated  in  the  earlier  work,  washed  leukocytes  are 
very  inconstant  with  respect  to  their  ability  to  phagocyte  oidio- 
mycetes.  The  addition  of  the  various  sera  mentioned  above  does 
not  help  matters.  It  was  thought  that  perhaps  the  manipulation 
to  which  they  were  subjected  injured  the  leukocytes  in  some  way 
and  an  effort  was  made  to  obviate  this  as  much  as  possible  by 
decreasing  the  number  of  washings  and  the  length  of  time  in  the 
centrifuge,  by  keeping  the  solutions  at  37°  C,  etc.,  but  without 
effect.  Once  in  a  while  such  leukocytes  ingested  organisms,  but, 
in  so  far  as  could  be  determined,  with  neither  rhyme  nor  reason. 
As  these  leukocytes  were  capable  of  ingesting  carmine  granules  as 
well  as  an  occasional  organism  it  appeared  that  they  had  not  lost 
their  powers  entirely;  why,  then,  this  failure  to  engulf  oidiomy- 
cetes  as  actively  as  the  leukocytes  in  the  fresh  exudate  ?  Several 
explanations  were  suggested.  The  technic  may  have  been  unsuited 
to  the  materials  with  which  the  work  was  being  done.  Savtchenko^'' 
found  that  the  mononuclear  cells  of  the  peritoneal  exudate  of 
guinea-pigs  have  a  tendency  to  clump  and  not  to  phagocyte 
after  centrifugation  in  citrate  solution.  Brisco^^  has  made  a  similar 
observation  with  respect  to  the  alveolar  cells  of  the  lung.  It  was 
noted  that  the  ingested  carmine  granules  were  much  smaller  than 
the  oidiomycetes.  Possibly  the  leukocytes  had  been  injured 
sufficiently  to  deprive  them  of  the  ability  to  envelop  such  giants 
as  the  yeasts  cells  but  still  retained  vitality  enough  to  engulf 
smaller  particles.  Again,  the  fresh  exudate  was  quite  viscid  and 
fibrin  threads  formed  within  it  in  a  very  short  time  after  the  mix- 
tures were  made.  Possibly  the  viscidity  of  the  medium  and  the 
fibrin  network  were  the  factors  which  enabled  the  leukocytes  to 
extend  themselves  over  the  organisms.  It  would  be  interesting, 
in  this  connection,  to  observe  the  effects  of  the  addition  of  a 
solution  of  gelatin  upon  the  phagocytic  activity  of  the  washed 
leukocytes.  This  might  also  be  the  key  to  the  eft"ects  of  dilution 
on  phagocytosis  in  the  fresh  exudate. 

(3)  The  proposition  that  phagocytosis  is  carried  on  in  the  fresh 
exudate  in  vitro  by  cells  of  the  same  type  as  those  which  are  respon- 


226  Contributions  to  Medical  Science 

sible  for  the  major  part  of  the  work  in  the  peritoneal  cav-ity  is 
borne  out  by  further  observations.  This  does  not  mean  that, 
either  within  the  peritoneal  cavity,  or  out  of  it,  all  the  work  is  done 
by  the  macrophages.  Such  is  not  the  case;  polymorphonuclear 
leukocytes  frequently  take  part  in  the  work,  and  sometimes  are 
the  only  leukocytes  present  in  the  plasmodial  masses.  They,  of 
course,  are  too  small  to  ingest  any  but  the  smallest  oidiomycetes. 
Nevertheless,  they  are  active  in  surrounding  and  ''hemming  in" 
the  organisms.  On  the  other  hand,  there  is  no  room  for  doubt 
that  the  bulk  of  the  work  is  done  by  the  macrophages. 

In  three  experiments,  normal  and  immune  serum  and  sodium 
citrate  solution  seemed  actually  to  inhibit  phagocytosis  in  fresh 
exudate,  while  the  leukocytes  in  the  salt-solution  dilution  of  the 
same  exudate  were  actively  phagocytic.  No  explanations  of  these 
results  have  suggested  themselves. 

Fresh  normal  rabbit  serum  was  mixed  with  guinea-pig  peritoneal 
exudate.  The  leukocytes  in  the  exudate  became  spherical  in  shape, 
and  were  strongly  agglutinated  within  30  minutes  at  37°  C.  The 
observations  were  not  carried  further.  Needless  to  say,  no  phago- 
cytosis occurred  in  such  preparations. 

These  experiments  seem  to  indicate  that  opsonins  are,  at  least, 
not  of  great  importance  in  the  phagocytosis  of  the  oidiomycetes 
used  in  this  work  by  the  leukocytes  of  the  peritoneal  exudates  of 
normal  and  immune  guinea-pigs. 

SUMMARY  AND   CONCLUSIONS. 

Oidiomycosis  in  the  guinea-pig,  following  intraperitoneal 
inoculation,  is  characterized,  in  fatal  cases  in  male  animals,  by  a 
gradually  developing  cachexia  accompanied  as  a  rule  by  a  steady 
loss  of  weight  which  is  especially  marked  during  the  last  three  or 
four  days  of  life  and  by  the  development,  in  pigs  of  400  gm.  or 
over,  of  palpable  nodules  in  the  testicles  in  from  three  to  seven 
days  after  inoculation.  In  males  weighing  400  gm.  or  less  the 
nodules  in  the  testicles  are  inconstant,  and,  when  present,  may  be 
difficult  to  palpate  owing  to  the  fact  that  they  occur  frequently 
about  the  upper  pole  of  the  testicle  and  about  the  neck  of  the 
scrotal  sac — points  which  may  be  difficult  to  feel  in  small  animals. 


Immunological  Reactions  of  Oidiomycosis  227 

The  appearance  of  palpable  nodules  in  the  skin  or  anterior  abdomi- 
nal wall  has  been  noted  but  may  be  avoided  in  a  great  measure 
by  rinsing  the  outside  of  the  needle  before  making  the  injection. 
In  female  guinea-pigs  symptoms  are  usually  entirely  lacking; 
they  survive  intraperitoneal  doses  which  kill  the  males,  a  point 
which  should  be  borne  in  mind  when  testing  the  pathogenicity  of 
yeasts  for  guinea-pigs.  The  scrotal  sac  of  the  male  pig  seems  to 
afford  a  locus  minoris  resistentiae  to  which  the  females  have  no 
counterpart.  The  reasons  for  the  lower  resistance  of  this  par- 
ticular corner  of  the  peritoneal-lined  cavity  are  not  apparent. 
Postmortem  examination  of  animals  dying  of  the  disease  reveals 
multiple  grayish  nodules  from  o.i  to  10  mm.  in  diameter  on  all 
peritoneal  surfaces.  All  such  nodules  contain  oidiomycetes,  as  may 
be  demonstrated  in  microscopic  sections  and  in  cultures.  Most 
nodules  have  softened  centers,  especially  the  large  nodules  which 
are  found  in  the  testicles,  in  which  oidiomycetes  and  great  numbers 
of  polymorphonuclear  leukocytes  occur.  The  lungs  and  liver  may 
also  present  small  areas  of  cellular  infiltration  about  oidiomycetes. 
There  are  no  constant  changes  in  the  various  organs. 

Recovery  from  an  infection  is  accompanied  by  a  low  grade  of  im- 
munity which  manifests  itself  in  a  somewhat  more  speedy  recovery 
from  subsequent  infections,  and  by  the  development  of  a  sUght  tem- 
perature for  a  few  days  immediately  following  reinoculations. 

Repeated  injections  of  an  "extract"  of  oidiomycetes  lead  to  the 
development  of  an  immunity  in  guinea-pigs  which  is  character- 
ized mainly  by  the  more  rapid  walling  off  of  organisms  injected 
into  the  peritoneal  cavity,  and  by  a  more  rapid  disappearance  of  the 
lesions  which  appear  in  the  testicles. 

The  sera  of  guinea-pigs  immunized  by  repeated  intraperitoneal 
injections  of  living  organisms  or  of  an  extract  of  organisms  develop 
precipitating  substances  against  the  '"extract"  in  from  50  to  79 
per  cent  of  the  cases,  which  may  be  manifest  in  a  dilution  as  high 
as  1 : 20.  Normal  sera  occasionally  precipitate  with  the  "extract" 
in  lower  dilutions.  Substances  which  will  agglutinate  oidiomy- 
cetes suspended  in  dilutions  of  the  sera  are  not  formed.  Specific 
amboceptors  also  do  not  appear  to  be  developed.  Opsonins  have 
not  been  demonstrated.     Prolonged  exposure  of  oidiomycetes  to 


2  28  Contributions  to  Medical  Science 

immune  or  normal  serum  at  37°  C.  seems  to  impair  the  vitality  of 
the  organisms — judging  by  their  diminished  power  of  growth  when 
transferred  to  suitable  media — as  compared  with  that  of  similarly 
tested  control  tubes  containing  organisms  suspended  in  physiologi- 
cal salt  solution.  The  reaction  of  anaphylaxis  may,  under  suit- 
able conditions,  be  obtained  with  the  "extract"  in  guinea-pigs. 
The  "extract"  is  toxic;  prolonged  administration  by  the  intra- 
peritoneal route  results  in  cachexia  and  death  usually  by  second- 
ary infection;  judiciously  used,  it  may  have  a  favorable  effect 
upon  the  course  of  an  oidiomycetic  infection. 

Oidiomycetes  injected  into  the  peritoneal  cavity  of  guinea-pigs 
are  rapidly  taken  by  leukocytes,  macrophages  principally,  which 
form  plasmodial  masses  and  become  attached  to  the  peritoneal 
surface  at  first  by  fibrinous,  later  by  fibrous,  adhesions,  the  mass 
of  cells  grouped  immediately  about  the  organisms  presently  assum- 
ing the  structure  of  typical  Langhans  giant  cells.  In  cases  ending 
in  recovery,  the  organisms  may  grow  for  a  time,  but  eventually 
degenerate  and  disappear;  the  nodule  within  which  they  were, 
being  absorbed  with  them.  In  fatal  cases  the  nodules  increase 
in  size  and  become  soft;  many  of  the  organisms  degenerate,  but 
others  multiply;  the  inclosing  giant  cell  disintegrates  and  there 
ensues  an  infiltration  of  polymorphonuclear  leukocytes  pari  passu 
with  the  enlargement  and  softening  of  the  nodule.  Sometimes 
the  organisms  are  surrounded  by  polymorphonuclear  cells  from  the 
beginning;  such  a  mass  may  in  turn  be  inclosed  by  layers  of  macro- 
phages. Just  what  such  a  condition  may  mean  as  regards  the 
prognosis  of  the  lesion  has  not  been  determined. 

The  organisms  do  not  tend  to  penetrate  the  peritoneal  surface. 
It  seems  probable  that  they  leave  the  peritoneal  cavity,  only  in 
case  there  has  been  actual  tearing  of  the  lining  tissues.  Oidio- 
mycetic nodules  were  described  in  the  liver  and  lungs  of  a  few 
animals.  The  belief  seems  to  be  justified  that  such  generalization 
of  the  infection  followed  the  introduction  of  the  organisms  into 
the  circulation  by  mechanical  means,  because  the  condition  occurred 
almost  solely  (there  were  two  exceptions)  in  animals  in  whose 
peritoneum  one  could  detect  tears,  as  in  the  animals  used  for  the 
study  of  phagocytosis  in  vivo. 


Immunological  Reactions  of  Oidiomycosis  229 

The  mode  of  defense  of  guinea-pigs  against  oidiomycetes  in- 
jected into  the  peritoneal  cavity  appears  to  consist,  firstly,  in 
phagocytosis  and  intracellular  digestion;  secondly,  in  a  walling 
off  and  encapsulation  of  the  phagocyted  organisms  by  connective 
tissue;  and,  thirdly,  upon  a  somewhat  ill-defined,  and  possibly 
questionable,  unfavorable  influence  of  the  serum  upon  the  vitality 
of  the  organism.  These  agencies  act  but  slowly;  oidiomycetes 
may  retain  their  power  to  grow  on  artificial  media  for  days,  and 
their  characteristic  staining  properties  for  weeks,  in  the  inflam- 
matory nodules  of  supposedly  immune  pigs.  Specific  antibodies 
are  but  poorly  developed. 

REFERENCES. 

1.  Metschnikoff.     Virchow's  Archiv,  1884,  96,  p.  177. 

2.  RiBBERT.     Der  Untergang  pathogener  Schimmelpilze  im  Korper,  Bonn,  1887. 

3.  Saxfelice.     Cenlralbl.  fur  BakL,  Abt.  i,  Orig.,  1896,  20,  p.  219. 

4.  Charrin  and  Ostrowsky.     Comptes  rendus  Soc.  de  Biol.,  1896,  48,  p.  743. 
S-  Roger.     Comptes  rendus  Soc.  de  Biol.,  1896,  48,  p.  728. 

6.  ScHATTENTROH.    Arcli.  fHf  Hyg.,  1896,  27,  p.  234. 

7.  GiLKiNET.     Arch,  de  med.  e.vper.,  1897,  9,  p.  881. 

8.  JONA.     Cenlralbl.  fUr  Bakt.,  Abt.  i,  Orig.,  1897,  21,  p.  147. 

9.  Obici.    Zeiglers  Beitrdge,  1898,  23,  p.  197. 

10.  Skchiwan.    Anti.  de  I'Inst.  Pasteur,  1899,  13,  p.  770. 

11.  Ricketts.    Jour.  Med.  Res.,  1901,  6,  p.  377. 

12.  Malvoz.    Ann.  de  la  soc.  med.-chir.  de  Liege,  1901,  quoted  from  Sanfelice  (15). 

13.  Malvoz.     Cenlralbl.  fiir  Bakt.,  Abt.  i,  Orig.,  1901,  29,  p.  668. 

14.  Savtchenko.     Ann.  de  I'Insl.  Pasteur,  1902,  16,  p.  106. 

15.  Sanfelice.     Cenlralbl.  fiir  Bakt.,  Abt.  i,  Orig.,  1902,  32,  p.  360. 

16.  Wlaeff.     Compt.  rendus  Soc.  de  Biol.,  1902,  54,  p.  412. 

17.  PoTRON.     Blastomycetes  dans  les  tissues.    These,  Nancy,  1903. 

18.  Fabozzi.     Cenlralbl.  fiir  Bakt.,  Abt.  i,  Ref.,  1906,  39  (Abstract),  p.  282, 

19.  Spiethoff.    Jahrbiicher  der  Hamb.  Staatskrankenanstalten,  1903-4,  9,  Heft  II, 
p.  167. 

20.  Bowen  and  Wolbach.    Jotir.  Med.  Res.,  1906,  15,  p.  167. 

21.  Christensen  and  Hektoen.    Jour.  Amer.  Med.  Assoc,  1906,  47,  p.  247. 

22.  Hamburger.    Jour.  Infect.  Dis.,  1907,  4,  p.  201. 

23.  Marco  del  Pont.     Bull,  de  I'Inst.  Pasteur,  1907,  5,  p.  46. 

24.  Brisco.    Jour.  Path,  and  Bad.,  1907,  12,  p.  66. 

25.  Brown.    Jour.  Amer.  Med.  Assoc,  1907,  48,  p.  743. 

26.  Hektoen.    Jour.  Amer.  Med.  Assoc,  1907,  49,  p.  1071. 

27.  Montgomery  and  Ormsby.    Archives  Int.  Med.,  1908,  2,  p.  i. 

28.  Rothe.     Deut.  med.  Wchnschr.,  1910,  36,  p.  30. 

29.  Widal,  Abrami,  Joltrain,  Brissaud,  et  Weill.     Ann.  de  I'Inst.  Pasteur,  1910, 
24,  p.  I. 


230  Contributions  to  Medical  Science 

EXPLANATION    OF    PLATE    i. 

Fig.  I  (at  top). — Inclusion  of  oidiomycetes  in  uninuclear  and  multinuclear 
cells  in  liver  (guinea-pig  53 — see  p.  195). 

Fig.  2  (in  center). — Leukocytic  rosette  from  peritoneal  exudate  (guinea-pig 
86 — see  p.  212). 

Fig.  3  (below). — Nodule  from  omentum  of  guinea-pig  showing  transformation 
of  rosettes  into  Langhans  giant  cells;  degenerative  change  in  oidiomycetes. 


PLATE   1. 


m     \^ 


^ 'I  a     *      e  "^ »  "^  *  ^  c   . 


9  ■%2-V'^.';,N^ 


LYMPHATOTOXIC    SERUM:     NOTES    ON   ITS    CONSTI- 
TUTION;   PRELIMINARY  EXPERIMENTS  BEARING 
ON    ITS    INFLUENCE    ON    EXPERIMENTAL 
INFECTIONS/ 

H.    T.    RrcKETTS. 
(From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

In  this  communication  no  attempt  will  be  made  to  summarize 
completely  the  literature  of  "leuko toxic"  or  " lymphato toxic " 
serum.  This  was  done  in  certain  aspects  in  a  recent  paper  by 
Flexner.^     A  few  important  data  may  be  given,  however. 

A  leukotoxic  serum  was  first  described  by  Metschnikofif  in 
1899.  Since  that  time  he  and  his  school  have  pubHshed  many 
papers  on  the  subject,  and  the  phenomenon  has  been  used  to 
support  the  phagocytic  theory  of  natural  immunity. 

Besredka  in  particular  has  contributed  important  work.  His 
findings,  which  have,  in  the  main,  been  supported  by  others,  are 
briefly  as  follows:  A  leukotoxic  serum  may  be  obtained  by  the 
immunization  of  one  animal  with  the  lymphatic  tissues  of  another, 
using  lymph  glands,  spleen,  bone-marrow,  or  pure  preparations 
of  leukocytes.  The  toxicity  of  the  serum  depends  on  the  number 
of  injections  made  and  on  the  animals  used;  the  guinea-pig  yields 
a  stronger  serum  when  treated  with  rabbit  tissue  than  vice  versa. 
Leukotoxic  sera  are  for  the  most  part  specific  for  the  cells  of  the 
animal  used  in  the  immunization,  but  this  is  not  without  excep- 
tions. All  leukotoxic  sera  are  more  or  less  hemolytic.  As  to 
toxicity,  it  is  possible  to  obtain  a  serum  from  a  rabbit  which  will 
kill  a  guinea-pig  in  a  dose  of  0.5  c.c.  in  a  few  hours.  He  also 
observed  that  intraperitoneal  injections  cause  a  marked  desquama- 
tion of  the  endothelium,  and  micro-organisms  may  then  the  more 
easily  wander  out  from  the  intestines  into  the  peritoneal  cavity. 

'  This  work  was  begun  in  the  laboratory  of  M.  Metschnikofif,  who  suggested  the  line  of  work.  The 
writer's  thanks  are  due  to  him  and  his  associates  for  very  numerous  kindnesses;  also  to  Mr.  Dick  at  the 
University  for  assistance  in  leukocyte  counts.    From  Trans.  Chic.  Path.  Soc,  1902,  5,  p.  178. 

» "The  Pathology  of  Lymphotoxic  and  Myelotoxic  Intoxication,"  Univ.  of  Penn.  Bulletin,  Novem- 
ber, 1902. 

231 


232  Contributions  to  Medical  Science 

When  an  intraperitoneal  injection  of  the  serum  is  made  there 
follows  rapidly  a  great  decrease  in  the  number  of  leukocytes  in 
the  exudate;  however,  an  extreme  hyperleukocytosis  supervenes, 
which  may  be  ten  times  in  excess  of  the  normal,  and  which  lasts 
for  a  number  of  days.  Further  injections  cause  a  further  increase 
of  leukocytes,  but  without  the  primary  leukopenia.  Besredka 
states  that  normal  rabbit  serum  produces  no  primary  hypoleu- 
kocytosis.  This  I  have  not  been  able  to  substantiate.  He  also 
found  that  small  doses  of  the  serum  given  subcutaneously  will 
produce  a  mild  general  hyperleukocytosis.  Normal  rabbit  serum 
had,  however,  the  same  effect.  Antileukotoxic  serum  prevented 
the  solution  of  the  leukocytes  by  the  leuko toxic  serum. 

The  agglutinating  value  of  the  serum  for  the  homologous 
leukocytes  is  also  a  recognized  phenomenon.  Both  the  fresh  and 
the  heated  serum  will  agglutinate.  Leukotoxic  sera  are  said  to 
be  less  stable  than  hemolytic  sera. 

Isaeff  found  that  with  the  resultant  hyperleukocytosis  the  resist- 
ance of  animals  to  experimental  infections  was  increased. 

Other  works  concerning  the  relation  of  "phagolysis"  to  immu- 
nity will  not  be  entered  on  here. 

The  serum  used  in  the  experiments  to  be  considered  was  pre- 
pared by  injecting  suspensions  of  the  mesenteric  lymph  glands  of 
guinea-pigs  into  rabbits.  The  glands  of  two  animals  were  used 
in  the  first  injection,  and  of  three  each  for  the  second  and  third 
injections.  The  suspensions,  made  by  grinding  the  tissue  with 
sterile  sand,  were  injected  seven  days  apart,  and  either  subcu- 
taneously or  into  the  peritoneal  cavity;  the  latter  method  gives 
a  somewhat  stronger  serum.  Blood  was  taken  from  the  rabbits  to 
obtain  the  serum  from  seven  to  ten  days  after  the  last  injection. 
The  serum  was  allowed  to  collect  spontaneously  at  a  somewhat 
lowered  temperature. 

Following  the  laws  of  the  formation  of  antibodies,  one  would 
expect  to  obtain  in  such  a  serum  antibodies  for  each  type  of  cell 
or  tissue  constituent  injected.  The  large  mass  of  cells  in  lymph 
glands  are,  of  course,  the  lymphoid  cells,  the  lymphocytes.  There 
are  in  addition  many  endotheUal  cells  and  a  small  amount  of 
circulating    blood.     In    accordance    with    these    expectations,    a 


Lymphatotoxic  Serum  233 

lymphotoxic  (or,  more  properly,  a  leukotoxic),  an  endotheliotoxic, 
and  a  hemotoxic  or  hemolytic  antibody  are  formed.  In  addition 
to  these,  an  agglutinating  and  a  precipitating  substance  seem  to 
be  formed. 

THE   LEUKOTOXIN. 

The  leukolytic  property  is  observed  when  the  fresh  or  reactivated 
serum  is  brought  in  contact  with  the  leukocytes  existing  normally 
in  the  peritoneal  or  pleural  exudate  of  the  guinea-pig,  or  when 
mixed  with  a  suspension  of  triturated  guinea-pig  lymph  gland. 
(Regardless  of  the  embryologic  origin  of  the  "endothelium" 
of  these  surfaces  it  seems  probable  that  they  have  certain  recep- 
tors identical  with  those  of  the  lymphatic  endothelium.)  So  far 
I  have  obtained  only  a  moderately  powerful  leukolytic  serum, 
judging  from  experiments  made  in  vitro.  It  is  possible  that  the 
actual  toxicity  of  the  serum  may  be  greater  than  such  experiments 
indicate.  One  part  of  serum  to  ten  parts  of  exudate  would  often 
cause  complete  solution  of  the  bodies  of  the  leukocytes,  the  nuclei 
and  granules  showing  much  greater  resistance;  commonly  one  to 
three  or  four  gave  more  positive  results.  The  leukocytes  when 
first  drawn  are  actively  motile  and  the  surfaces  are  rough  with 
many  small  pseudopodia.  Kept  at  the  proper  temperature, 
this  may  keep  up  for  several  hours.  Almost  immediately,  however, 
when  mixed  with  leukotoxic  serum  pseudopodia  cease  to  form, 
and  the  surface  of  the  cell  becomes  perfectly  smooth  in  a  few 
minutes.  The  cell  next  enlarges,  the  protoplasm  becomes  clear, 
the  nucleus  shows  more  distinctly,  and  the  cell  appears  to  be  in- 
closed by  a  thin  clear  membrane.  The  swelling  continues,  one 
portion  of  the  surface  may  balloon  out,  the  nucleus  often  becomes 
eccentric,  and  the  protoplasmic  granules  group  themselves  about 
the  nucleus.  Solution  may  not  extend  beyond  this  point,  or  if 
there  be  a  sufficient  amount  of  antibody  and  complement  the  ceil 
membrane  becomes  dissolved  and  the  granules  are  set  free.  While 
the  granules  are  not  unaffected  they  have  about  the  same  resist- 
ance to  solution  as  the  nucleus.  All  leukocytes  are  not  attacked 
equally;  the  first  to  show  changes  are  the  large  finely  granular  cells, 
the  macrophages;  the  polymorphonuclears  are  attacked  at  about 
the  same  time.     The  most  resistant  are  the  small  mononuclear 


234  Contributions  to  Medical  Science 

cells,  the  microphages,  which  contain  coarser  granules  than  the 
macrophages.  With  only  a  moderately  toxic  serum  the  micro- 
phages may  become  swollen  as  much  as  thirty  minutes  to  an  hour 
later  than  the  macrophages  or  the  polynuclears. 

A  particular  experiment  showed  that  freezing  for  one  and  one- 
half  to  two  hours  did  not  destroy  the  lytic  property  of  the  serum 
for  leukocytes;  a  temperature  of  55°  C.  for  thirty  minutes  does, 
and  the  serum  can  be  only  partially  reactivated  by  the  use  of  fresh 
serum. 

THE   ENDOTHELIOTOXIN. 

This  action  is  manifested  in  intraperitoneal  and  intrapleural 
injections  of  the  fresh  serum  by  the  production  of  a  marked  des- 
quamation of  the  endothelium.  Normal  rabbit  serum  produced 
no  such  desquamation  or  a  trifling  amount.  The  cells  are  seen 
singly  and  in  shreds.  Furthermore,  changes  in  shape  occur  after 
the  desquamation  of  the  cells.  A  third  point  in  evidence  is  the 
formation  of  a  marked  hematoma  at  the  point  of  a  subcutaneous 
injection.  The  peritoneal  desquamation  occurs  very  rapidly, 
within  fifteen  minutes  following  the  injection,  and  progresses  for 
a  half  to  one  hour.  From  this  time  on  the  number  found  decreases 
steadily.  The  reason  is  not  clear,  but  possibly  depends  on  the 
agglutination  of  the  cells  and  their  adhering  to  the  parietes.  After 
desquamation  the  changes  that  occur  in  the  cells  are  somewhat 
insignificant.  They  do  not  become  spherical,  but  are  somewhat 
swollen  laterally,  and  irregularities  in  the  surface  are  smoothed 
out.  In  the  unaltered  cells,  as  they  appear  when  first  desquamated, 
the  borders  are  irregularly  rectangular  and  the  edges  refract  the 
light  brilliantly;  this  is  probably  due  in  part  to  adhering  cement 
substance.  After  being  subjected,  in  vitro,  for  about  fifteen 
minutes  to  the  action  of  the  immune  serum  these  refracting  edges 
become  less  distinct  and  eventually  few  of  them  can  be  seen. 
This  is  seemingly  due  to  a  solution  of  the  adhering  cement  sub- 
stance as  well  as  to  alteration  in  the  shape  of  the  cell.  The  effect 
on  the  cement  substance  may  also  play  a  part  in  the  desquama- 
tion. It  is  thus  seen  that  the  visible  effects  on  the  endothelial 
cells  are  much  less  than  on  the  leukocytes. 

The  hematoma  which  follows  subcutaneous  injection  develops 


Lymphatotoxic  Serum  235 

in  the  course  of  one  to  two  hours,  i.e.,  the  fluid  at  that  time  is 
found  to  be  hemorrhagic.  The  amount  of  blood  increases  during 
the  first  twenty-four  hours,  and  it  is  diluted  with  a  large  amount 
of  lymph.  Many  leukocytes  are  found  in  the  fluid  in  incomplete 
agglutination;  a  few  of  these  show  moderate  lytic  changes.  The 
tumor  subsides  in  the  course  of  a  week  or  ten  days.  Histologically 
(tissue  removed  on  the  second  day)  there  is  an  enormous  edema 
and  vast  fields  of  leukocytes,  particularly  polymorphonuclear. 
The  fibrous  tissue  cells  are  in  active  proliferation  and  show  mitoses, 
and  the  collagenous  and  muscular  tissues  are  degenerated.  The 
endothehal  cells  of  blood-vessels  and  lymph  spaces  have  increased, 
and  are  often  greatly  swollen,  projecting  prominently  into  the 
lumen.  It  was  not  possible  to  demonstrate  vascular  rhexis  histo- 
logically. The  overlying  skin  becomes  necrotic  and  can  easily  be 
wiped  off. 

THE  HEMOLYSIN. 

The  hemolytic  value  of  the  immune  serum  is  not  greatly  above 
that  of  normal  rabbit  serum  for  guinea-pig  corpuscles;  in  a  care- 
fully conducted  experiment,  0.05  c.c.  of  the  former  completely 
dissolved  a  given  quantity  (0.4  c.c.)  of  a  5  per  cent  suspension  of 
guinea-pig  corpuscles  in  0.85  per  cent  salt  solution,  while  o.i 
c.c.  of  the  latter  was  required. 

THE   LEUKO-AGGLUTININ. 

The  agglutination  appears  in  a  few  minutes  after  the  mixture 
is  made  in  vitro,  and  also  occurs  with  equal  rapidity  in  intraperi- 
toneal injections.  That  the  agglutinating  property  is  distinct 
from  the  lytic  is  shown  by  the  fact  that  when  deprived  of  the 
complement  by  heating,  or  as  a  result  of  standing,  agglutination 
will  still  occur.  Normal  rabbit  serum  has  slight  agglutinating 
and  lytic  properties  for  guinea-pig  leukocytes,  but  much  less  than 
that  obtained  by  immunization. 

THE   PRECIPITIN. 

Precipitation  is  observed  when  one  makes  the  mixture  used  for 
studying  leukolysis.  A  control  of  normal  rabbit  serum  and  guinea- 
pig  peritoneal  exudate  shows  a  barely  perceptible  amount  of  the 


236  Contributions  to  Medical  Science 

precipitate.  Bouillon  and  0.85  per  cent  salt  solution  with  the 
exudate  show  no  precipitate. 

It  is  then  evident  that  we  have  to  deal  with  a  complex  serum, 
one  not  purely  leukotoxic  or  lymphotoxic,  and  it  is  probable  that 
this  must  be  taken  into  consideration  in  drawing  conclusions 
concerning  the  effect  of  the  serum  on  experimental  infections. 

The  course  of  peritoneal  leukocytosis  following  intraperitoneal 
injections  of  the  serum  has  been  observed  and  certain  findings  of  pre- 
vious workers  corroborated  (especially  Metschnikojff  and  his  school). 
Controls  were  made  with  normal  rabbit  serum  and  bouillon.  It 
is  a  generally  known  fact  that  many  substances  when  introduced 
into  the  peritoneal  cavity  cause  an  influx  of  leukocytes.  With 
these  substances  there  is  usually  a  primary  leukopenia,  hyper- 
leukocytosis  supervening  in  from  one  to  two  hours  and  increasing 
for  about  twenty-four  hours.  It  then  gradually  decreases  during 
the  course  of  several  days.  The  estimation  of  the  primary  leuko- 
penia is  subject  to  no  small  error,  owing  to  the  dilution  of  the 
exudate  by  the  fluid  injected.  Since  there  appears  to  be  normally 
not  more  than  0.5  c.c.  of  fluid  in  the  abdominal  cavity  of  a  guinea- 
pig,  it  is  clear  that  the  injection  of  3  c.c.  of  fluid  would  produce  in 
itself  an  apparent  leukopenia,  and  it  is  even  possible  that  this 
may  constitute  the  entire  leukopenia  described.  Another  error 
is  caused  by  the  agglutination  of  the  leukocytes  on  the  peritoneal 
walls  when  a  foreign  substance  is  injected.  Concerning  the  even- 
tual hyperleukocytosis  there  can,  however,  be  no  question.  Often 
it  was  not  possible  to  follow  out  the  counts  more  than  twenty-four 
hours  when  the  leukotoxic  serum  was  injected,  on  account  of  a 
marked  viscosity  which  developed  in  the  exudate.  It  is  to  be  noted 
that  with  all  three  solutions  there  followed  a  primary  leukopenia 
which  was  in  part,  at  least,  apparent  for  the  reason  mentioned. 
The  hyperleukocytosis  was  usually  most  marked  following  the 
leukotoxic  serum,  but  both  bouillon  and  normal  rabbit  serum 
often  gave  a  vast  increase.  In  the  stage  of  hyperleukocytosis  the 
polymorphonuclears  are  in  excess  of  all  other  cells,  while  in  the 
natural  condition  they  are  very  limited  in  number.  It  will  be 
remembered  that  the  same  cell  constitutes  the  mass  of  white  cells 
found  in  the  subcutaneous  exudate  also.     It  was  mentioned  that 


Lymphatotoxic  Serum  237 

the  peritoneal  exudate  soon  becomes  thick  and  viscous.  Pieral- 
lini  attributes  this  condition  to  fibrin  formation  due  to  the  disinte- 
gration of  the  leukocytes  (phagolysis) ,  basing  his  conclusion  on 
the  positive  reaction  to  Weigert's  fibrin  stain. 

The  following  is  an  example  of  the  course  of  leukocytosis  in 
two  cases  following  an  intraperitoneal  injection  of  leukotoxic 
serum,  with  a  control  of  normal  rabbit  serum: 


Leukotoxic  serum: 

A. 

Before 
Inj. 

14,400 

Extreme 
Leukopenia 

266 

Extreme 
Hyperleuk. 

282,700 

B. 

9,000 

1,532 

662,000 

Normal  senmi: 

A. 
B. 

14,400 
17,200 

1,200 
1,266 

72,000 
68,000 

The  serum  used  in  these  experiments  was  fairly  powerful. 
This  probably  accounts  for  the  extreme  hyperleukocytosis.  More 
recent  sera  have  provoked  lower  grades  of  hyperleukocytosis. 

That  the  secondary  hyperleukocytosis  is  not  a  specific  result 
of  the  injection  of  leukotoxic  serum  is  shown  in  two  cases  where 
two  c.c.  of  peptone  bouillon  were  injected. 


Before 
Inj. 

E.xtreme 
Leukopenia 

Extreme 
Hyperleuk 

A. 

59,000 

2,400 

267,000 

B. 

34,075 

3,200 

250,000 

Here  the  leukopenia  seems  real.  The  hyperleukocytosis  is 
extreme,  and  in  this  connection  it  is  perhaps  of  significance  that 
Delezenne  observed  that  a  solution  of  Witte's  peptone  produced 
a  marked  degree  of  leukocytosis  in  vitro.  It  seems  probable  that, 
within  certain  limits,  the  greater  the  toxicity  or  leukolytic  power 
of  the  substance  injected  the  greater  the  resultant  hyperleukocy- 
tosis;   this,  however,  needs  further  corroboration. 

The  course  of  peritoneal  and  vascular  leukocytosis  has  been 
followed  after  the  subcutaneous  injection  of  2  c.c.  of  leukotoxic 
serum.  There  was  in  neither  fluid  a  primary  leukopenia,  but 
almost  immediately  a  moderate  rise  in  each.  In  twelve  hours  the 
number  began  to  decrease  almost  uniformly  in  the  two  fluids; 
as  this  decrease  is  synchronous  with  the  pronounced  development 
of  the  subcutaneous  hema-leukocytoma  it  may  be  supposed  that 
a  vast  number  of  leukocytes  was  abstracted  from  the  blood  and 


238  Contributions  to  Medical  Science 

lymph  to  swell  those  at  the  point  of  injection.  After  twenty-four 
hours  this  began  to  be  compensated  for,  a  rise  occurring  in  both 
the  blood  and  peritoneal  exudate.  One  may  consider  this  an  illus- 
tration of  excessive  repair  after  an  injury  (Weigert). 

Now  follows  a  brief  consideration  of  the  relations  of  these 
artificially  induced  h>'po-  and  h}^er-leukocytoses  to  experimental 
infections.  The  series  was  begun  with  the  cholera  and  typhoid 
organisms,  but  owing  to  the  great  number  of  animals  required  the 
cholera  vibrio  was  abandoned  after  some  preliminary  experiments 
which  showed  that  it  conducted  itself  similarly  to  the  typhoid 
bacillus. 

A  constant  method  of  growing  the  organisms  was  necessary 
to  obtain  uniformity  in  doses  and  virulence.  For  a  time  uniform 
agar  surfaces  were  employed,  but  this  was  later  abandoned  for 
a  constant  amount  of  peptone  bouillon.  Twenty-four-hour  cul- 
tures were  used. 

The  work  falls  naturally  into  two  aspects:  First,  in  the  primary 
peritoneal  leukopenia,  is  the  resistance  of  the  animal  to  peritoneal 
infections  increased?  Second,  during  the  stage  of  hyperleukocy- 
tosis  is  the  resistance  increased? 

For  the  first  the  procedure  was  as  follows:  The  serum  was 
injected  at  a  given  time  and  at  the  height  of  leukopenia  a  sublethal 
dose  of  the  bacterium  followed.  Three  controls  were  made  in 
typical  experiments,  one  to  insure  that  the  dose  of  culture  was  not 
fatal,  a  second  to  show  that  the  serum  would  not  kill  the  animal, 
and  a  third  with  the  same  amount  of  normal  rabbit  serum  and 
culture  used  in  the  experiment  animal.  After  many  repetitions 
the  following  result  was  finally  reached:  Leukotoxic  immune 
serum  (3  c.c.  of  the  heated  serum)  decreases  the  natural  immunity 
of  the  guinea-pig  for  typhoid  and  cholera,  when  the  culture  is 
injected  one  hour  after  the  serum,  so  that  one-half  the  M.L.D.  is 
fatal,  both  injections  being  made  into  the  peritoneal  cavity.  It 
developed  that  normal  rabbit  serum  had  a  similar  but  less  powerful 
effect  when  heated,  but  when  the  fresh  serum  was  used  the  ani- 
mals would  withstand  much  more  than  the  M.L.D.  This  is 
attributable  to  the  marked  bactericidal  property  of  fresh  rabbit 
serum  for  the  typhoid  bacillus.     The  exact  details  of  the  experi- 


Lymphatotoxic  Serum  239 

ments  will  be  given  later,  when  other  work  in  the  same  direction  is 
completed. 

In  dealing  with  the  second  part  of  the  question,  animals  were 
injected  with  a  given  dose  of  the  serum,  and  twenty-four  hours 
later  the  resistance  tested  as  before.  This  procedure  resulted 
in  immunity  against  about  twice  the  M.L.D.  of  the  typhoid  bacillus. 
(The  cholera  organism  was  abandoned  at  this  point.)  All  requisite 
controls  were  likewise  used  in  this  series.  It  was  also  brought  out 
that  normal  rabbit  serum  had  the  same  protective  value  as  the 
leukotoxic  serum  used  in  this  series.  When  one  considers  the 
difference  in  the  degrees  of  leukocytosis  produced  by  the  two  it 
becomes  apparent  that  the  normal  serum  had  even  greater  pro- 
tective value  than  the  leukotoxic  serum.  That  is  to  say,  75,000 
leukocytes  in  the  former  case  would  be  able  to  save  the  animal 
from  as  great  a  dose  of  bacteria  as  300,000  or  even  600,000  in  the 
latter  (assuming,  of  course,  that  the  leukocytes  are  really  the 
protective  agents).  This  may  be  explained  by  the  supposition 
that  the  leukocytes  called  out  by  the  immune  serum  are  still  in 
a  debilitated  state  as  the  result  of  the  lytic  effect  of  the  serum,  and 
consequently  had  a  lower  phagocytic  index. 

Some  preliminary  work  has  shown  that  peptone  bouillon  has 
similar  effects  to  leukolytic  and  normal  rabbit  serum  in  the  respects 
under  consideration,  although  to  a  less  degree. 

From  these  experiments  it  seems  probable  that  many  substances 
may  be  found  which  by  a  deleterious  influence  on  the  leukocytes 
(phagolysis,  or  otherwise  engaging  them)  are  able  to  decrease  the 
resistance  of  an  animal  to  certain  bacterial  infections  at  definite 
points  of  inoculation;  or  by  varying  the  mode  of  experimentation, 
the  same  substances  may  be  the  means  of  increasing  the  resistance 
to  the  same  infections,  the  result  depending  in  this  case  on  an 
increase  in  the  number  of  leukocytes,  or  of  complement  (Wasser- 
mann),  or  perhaps  both,  at  the  point  of  injection. 

The  participation  of  the  endothelium  in  these  processes  is 
undetermined  but  seems  probable  where  the  resistance  is  decreased 
as  indicated. 


PRELIMINARY  REPORT  ON  THE  ACTION  OF  NEURO- 
TOXIC SERUM/ 

H.     T.     RiCKETTS     AND     T.     ROTHSTEIN. 
(From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

We  shall  not  discuss  the  literature  of  neurotoxic  serum  in  this 
communication  further  than  to  mention  that  Delezenne,  Centanni, 
and  Enriquez  and  Sicard  have  produced  such  sera  by  immunization. 
Brief  re\'iews  of  these  works  are  made  by  Hektoen  in  Progressive 
Medicine,  March,  1902,  pp.  283,  284,  where  the  original  references 
are  given. 

We  have  immunized  rabbits  with  nervous  tissue  of  guinea-pigs, 
and  in  three  series;  using  in  the  first  the  cerebrum,  in  the  second 
the  cerebellum,  and  in  the  third  the  spinal  cord.  This  has  been 
satisfactory  so  far  as  the  strength  of  the  resulting  sera  is  concerned, 
although  Delezenne  found  that  injections  of  guinea-pigs  with 
nervous  tissue  of  rabbits  did  not  yield  a  strong  serum.  He  attrib- 
uted this  to  the  close  relationship  of  the  two  animals.  However, 
the  fact  that  rabbit's  serum  is  normally  more  toxic  to  the  corpuscles 
of  the  guinea-pig  than  the  serum  of  the  latter  animal  for  rabbit's 
corpuscles  may  suggest  an  explanation  of  our  better  success  in 
following  the  opposite  procedure  of  that  of  Delezenne. 

Large  amounts  of  tissue  were  used  in  immunization,  and  usually 
the  serum  was  collected  ten  days  after  the  third  injection. 

The  hemolytic  power  of  the  serum  for  erythrocytes  of  guinea- 
pigs  was  about  ten  times  as  great  as  normal  rabbit  serum  (for  the 
lower  limit),  in  one  experiment.  This  increase  in  hemolytic 
amboceptors  is,  no  doubt,  due  in  part  to  blood  included  in  the 
injected  mass,  but  it  is  also  possible  that  proper  receptors  existed 
to  some  extent  in  the  brain  cells  and  cerebral  fluid.  The  quantities 
of  neurotoxic  serum  injected  into  guinea-pigs  did  not  cause  any 
marked  hemolysis,  as  distinct  hemoglobinuria  was  not  observed  in 
fatal  cases.  The  finding  of  pigmented  cells  in  the  spleens  of  such 
animals  indicates,  however,  that  hemolysis  in  vivo  does  occur. 

When  small  amounts  of  the  immune  serum   (0.05-0.1   c.c.) 

»  Trans.  Chic.  Path.  Soc,  1903,  $,  p.  207. 

240 


The  Action  of  Neurotoxic  Serum  241 

are  added  to  0.5  c.c.  of  guinea-pig  serum  a  distinct  precipitate  is 
produced.  A  serum  sLx  weeks  old  and  not  recomplemented  gives 
the  reaction.  Normal  rabbit  serum  has  this  same  power,  but  to 
a  very  limited  degree.    Therefore  a  precipitin  is  formed. 

In  the  injected  animals  hemorrhages  occur  commonly  in  the 
meninges  and  lungs,  and  in  one  animal  they  were  found  in  the 
muscles,  hence  the  active  serum  is  probably  also  endothelio toxic. 
Intraperitoneal  injections  cause  a  marked  desquamation  of  endothe- 
lium. 

It  has  been  impossible,  so  far,  to  detect  solution  of  nervous 
tissue  in  test-tube  experiments,  or  in  microscopic  preparations. 
But  it  seems  probable  that  the  value  of  the  serum  in  antibodies  may 
be  estimated  approximately  by  the  precipitation  of  an  emulsion 
or  an  extract  of  nervous  tissue.  A  cerebral  hemisphere  of  a  guinea- 
pig  was  thoroughly  ground  with  sand,  intimately  emulsified  in 
20  c.c.  of  distilled  water,  and  passed  through  a  fine  wire  cloth. 
Half  of  this  was  filtered  through  two  layers  of  ordinary  filter  paper, 
and  the  fluids  used  in  precipitation  experiments.  The  filtered 
portion,  even  after  being  passed  through  the  paper  several  times, 
was  uniformly  cloudy-opalescent ;  microscopic  examination  showed 
the  presence  of  innumerable  fine  granules.  So  it  was  not  purely 
an  extract,  but  an  extract  plus  minute  portions  of  nervous  tissue. 
From  one  to  ten  drops  of  an  old  serum  were  added  to  small  tubes, 
each  containing  o .  5  c.c.  of  one  of  the  fluids.  It  was  found  that  two 
to  four  drops  of  either  the  anticerebral  or  the  anticerebellar  serum 
would  completely  clear  the  unfiltered  emulsion,  causing  a  dense, 
coherent  precipitate.  The  same  result  occurred  with  the  filtered 
portion,  naturally  with  a  smaller  amount  of  precipitate,  and  for 
some  reason  the  overlying  fluid  was  not  so  thoroughly  cleared  as 
in  the  unfiltered  portion.  This  experiment  indicates  that  anti- 
cerebral  and  anticerebellar  sera  are  not  specific  for  the  homolo- 
gous parts  of  the  nervous  system.  That  this  test  may  be  an 
indication  of  the  amount  of  antibodies  present  is  shown  by  the 
fact  that  a  serum  taken  after  two  injections  produced  a  consider- 
ably smaller  precipitate  in  the  same  solutions,  and  this  serum  was 
also  less  toxic  when  injected.  It  must  be  mentioned  that  normal 
rabbit  serum  has  similar  precipitating  power,  but  to  so  small  a 


242  Contributions  to  Medical  Science 

degree  that  it  is  easy  to  avoid  error.  A  definite  statement  as  to 
whether  this  action  is  purely  agglutinating,  or  mixed  agglutinating 
and  precipitating,  cannot  be  made  until  the  test  is  apphed  suc- 
cessfully to  an  extract  free  from  morphological  elements. 

The  neurotoxic  serum  has  been  injected  into  the  vessels,  the 
abdomen,  and  beneath  the  skin  of  guinea-pigs.  Although  symp- 
toms are  produced  by  the  last  two  methods,  they  have  not  been 
used  extensively,  as  much  less  serum  is  required  in  the  case  of 
intravascular  injections. 

The  following  brief  reports  of  experiments  illustrate  the  symp- 
toms which  may  follow  intravascular  injections. 

Cerebral  neurotoxin,  guinea-pig  i. — 2  c.c.  of  active  serum  injected 
in  left  carotid,  toward  brain.  Retarded  and  labored  respiration, 
at  first  deep,  then  superficial,  ensued  almost  immediately.  No 
convulsions,  profuse  urination;  death  in  3  to  5  minutes.  2  c.c. 
of  normal  rabbit  serum  injected  similarly  produced  only  slight 
muscular  twitchings,  which  soon  ceased  (Cerebr.  Nt.,  Control, 
guinea-pig  5).  Autopsy  of  guinea-pig  i  showed  only  a  slightly 
blood-stained  fluid  covering  the  brain  and  minute  intramuscular 
hemorrhages. 

Cerebral  neurotoxin,  guinea-pig  4. — 2  c.c.  active  serum  injected 
in  right  jugular  toward  heart.  In  3  to  5  minutes  appeared  mori- 
bund, the  only  sign  of  life  being  light  and  slow  respiration.  Passive, 
limp,  no  convulsions.  Urinated.  Pain  sense  present.  Eyes 
staring  and  slightly  bulging,  but  muscles  not  paralyzed.  In  two 
hours  began  to  recover  slowly  from  the  marked  prostration.  In 
eight  hours  was  able  to  move  about  slowly  and  with  difl&culty. 
Would  lie  down  almost  continuously  in  the  cage.  In  ten  days 
paralysis  of  the  posterior  limbs  had  developed,  the  fore  legs  were 
weakened,  and  emaciation  was  marked.  Died  in  fourteen  days 
with  no  new  symptoms.  The  patellar  reflexes  were  not  lost  at 
any  time  (3  c.c.  of  normal  rabbit  serum  injected  in  the  jugular 
produces  no  symptoms). 

At  the  autopsy  of  this  animal  the  frontal  and  sphenoidal  lobes 
were  very  soft  and  glistening;  no  congestion  or  edema.  The 
erector  spinae  muscles  and  the  posterior  muscles  of  the  thighs  were 
pale  and  waxy  in  appearance. 


The  Action  of  Neurotoxic  Serum  243 

Spinal  neurotoxin,  guinea-pig  i. — 2  c.c.  of  this  serum  injected 
in  the  right  carotid  was  followed  by  prolonged  spasms,  curvature 
of  the  body  toward  the  injected  side,  exophthalmia,  lachr>Tnation, 
and  transient  paresis  of  the  left  side.  The  deformity,  paresis, 
and  convulsions  ceased  after  four  hours.  Dyspnea  then  developed 
and  death  occurred  in  five  hours. 

Smaller  amounts  of  cerebral  and  spinal  sera  produce  convul- 
sions which  last  a  short  time,  and  prostration  and  emaciation  of 
several  days'  duration. 

Anticerebellar  serum  produced  convulsions,  prostration,  lachry- 
mation,  and  emaciation. 

In  animals  dying  a  few  hours  after  injection,  hemorrhages  have 
been  found  in  the  brain  and  meninges,  lungs  and  muscles. 

Changes  in  the  central  nervous  system  (Dr.  Rothstein). — The 
anatomical  changes  in  the  central  nervous  system  may  be  summed 
up  in  two  words:  hemorrhage  and  degeneration,  i.e.,  degeneration 
of  the  ganglion  cells. 

To  facilitate  description  the  animals  may  be  divided  into  three 
groups :  first,  those  which  died  one-half  to  one  hour  after  the  injec- 
tion; second,  those  which  died  three  and  one-half  to  four  and  one- 
half  hours  after;  and  third,  the  one  animal  which  died  fourteen 
days  after  the  injection  of  the  neurotoxic  serum. 

In  the  animals  of  the  first  group  were  found  multiple  pial  and 
subpial  hemorrhages  in  the  cerebrum,  cerebellum,  and  spinal  cord. 
Blood  extravasates  also  existed  in  the  gray  matter,  especially 
around  the  central  canal  and  in  the  anterior  horns  of  the  spinal 
cord.  In  one  animal  a  hemorrhage  had  completely  destroyed  the 
anterior  horn  on  one  side  in  the  cervical  region.  Not  a  trace  of 
the  central  canal  could  be  detected,  and  the  hemorrhage  extended 
in  the  central  part  of  the  cord  up  into  the  fourth  ventricle.  Puncti- 
form,  as  well  as  more  extensive  hemorrhages  existed  in  the  tissue 
beneath  the  ependyma,  and  the  ependyma  was  in  places  entirely 
destroyed.  The  ganglion  cells,  on  the  other  hand,  were  only 
slightly  altered.  The  great  majority  of  them  might  be  used  to 
demonstrate  the  normal  structure  of  the  nervous  cells.  In  a  few 
places,  however,  can  be  observed  a  sUght  but  distinct  chromatolysis. 

The  preparations  from  the  second  group  of  animals  present  a 


244  Contributions  to  Medical  Science 

vastly  different  picture.  The  hemorrhages  are  few,  small  in  size, 
and  only  found  in  the  pia  mater.  The  ganglion  cells,  on  the  other 
hand,  are  all  more  or  less  changed,  and,  as  a  general  rule,  it  may  be 
said  that  the  larger  the  cells  the  greater  are  the  changes  they 
present.  The  cells  in  the  spinal  root  ganglia  are  an  exception  to 
this  rule,  being  markedly  less  changed  than  the  other  kinds  of 
nervous  cells.  In  the  spinal  cord  only  a  few  cells  are  found  in 
which  the  Nissl  bodies  can  be  distinguished,  but  the  tigroid  sub- 
stance, even  in  these  cells,  is  less  in  amount  and  without  the  distinct 
arrangement  of  the  normal  cell.  In  some  cells  are  found  only 
traces  of  the  Nissl  bodies  and  in  still  others  they  have  entirely 
disappeared,  the  cells  being  lightly  and  diffusely  stained.  But 
not  only  the  tigroid  substance  is  altered.  Many  of  the  cells, 
especially  the  great  motor  cells,  show  vacuolization,  and  sometimes 
the  nucleus  has  lost  its  outlines.  Still  greater  changes  are  found  in 
a  considerable  number  of  cells,  the  protoplasm  being  in  a  state  of 
dissolution,  presenting  a  picture  that  reminds  one  of  coagula- 
tion necrosis. 

The  ganglion  cells  of  the  cerebellum  and  cerebrum  also  show 
alterations  to  a  greater  or  less  degree. 

In  the  preparations  from  the  third  group  were  found  still 
fewer  hemorrhages  than  in  the  second  group.  The  chroma- 
tolysis  may  be  said,  as  a  rule,  to  be  more  general.  In  nearly  all  the 
cells  the  Nissl  bodies  have  entirely  disappeared;  but  other  changes 
of  the  protoplasm  are  not  so  great  as  in  the  second  group.  The 
cells  look  homogeneous,  staining  slightly  and  dift'usely.  Vacuo- 
lization is  not  found  frequently,  and  seldom  is  a  cell  seen  that 
might  be  said  to  be  in  a  state  of  necrosis.  Usually  the  nucleus 
appears  normal.  The  chromatolysis  extends  over  the  whole 
central  nervous  system. 


THE  REDUCTION  OF  METHYLENE  BLUE  BY  NERVOUS 

TISSUE/ 

H.     T.     RiCKETTS. 

(From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

A  number  of  years  ago  Ehrlich  pointed  out  the  ability  of  living 
cells  to  decolorize  (i.e.,  reduce)  solutions  of  methylene  blue.  Later, 
Neisser  and  Wechsberg^  devised  an  application  of  this  phenomenon 
to  determine  the  cytocidal  effect  of  some  cellular  poisons  upon 
leukocytes.  They  found  that  living  leukocytes  were  able  to 
reduce  the  dye  quantitatively,  but  when  they  were  killed  by 
heat,  alcohol,  an  immune  leuko toxic  serum,  or  staphylococcus 
"leukocidin,"  they  lost  this  power.  Other  cells,  when  fresh  (sper- 
matozoa, pancreas,  kidney),  also  reduced  the  dye,  but  not  after 
treatment  with  an  injurious  agent  (alcohol).  Living  micro- 
organisms had  a  similar  effect,  which  was  lost  on  treating  them 
with  a  bactericidal  substance.  Ferments  and  bacterial  toxins  had 
no  reducing  power. 

I  attempted  to  use  this  method  in  determining  the  toxic  effect 
of  an  immune  neurotoxic  serum  upon  nervous  tissue.  The  serum 
was  prepared  by  immunizing  rabbits  and  geese  with  the  central 
nervous  system  of  guinea-pigs,  and  was  generally  of  such  strength 
that  2.0  c.c.  intravenously,  0.5-1  c.c.  in  the  carotid,  or  0.01-0.5 
c.c.  intracerebrally  caused  the  death  of  a  guinea-pig  in  two  to  twelve 
hours.  The  histological  examination  of  tissues  as  reported  by 
Ricketts  and  Rothstein^  shows  that  this  serum  produces,  intra 
vitam,  profound  changes  in  the  nerve  cells,  while  the  effects  may 
extend  to  other  tissues  as  well.  In  view  of  the  results  of  Neisser 
and  Wechsberg,  it  was  expected  that  this  "bioscopic"  method 
might  be  applied  here  to  advantage. 

Neisser  and  Wechsberg  placed  their  mixture  of  cells,  toxin, 
and  methylene  blue  in  small  test-tubes,  and  covered  the  fluid  with 
liquid  paraffin  to  exclude  the  air,  this  being  necessary  for  the 
accomplishment  of  reduction.     I  have  found  more  convenient  a 

■  From  Jour.  Infect.  Dis.,  1904,  i,  p.  590. 

'  Miinch.  med.  Wchnschr.,  1900,  47,  p.  1261;   Zlschr.  f.  Hyg.,  1901,  36,  p.  330. 

J  Trans.  Chic.  Path.  Soc,  1903,  $,  p.  207. 

245 


246  Contributions  to  Medical  Science 

tube,  suggested  by  Professor  Hektoen,  of  4-5  mm.  in  diameter, 
drawn  to  a  fine  point  at  either  end,  which  can  be  sealed  in  the 
flame  after  the  tube  is  filled. 

For  the  first  experiments  an  emulsion  of  a  guinea-pig  brain  in 
30  c.c.  of  0.85  per  cent  NaCl  was  used,  the  tissue  being  ground 
fine  with  quartz  sand,  from  which  it  was  decanted;  later  a  5  per 
cent  emulsion  was  used  uniformly.  Aseptic  precautions  were 
used  throughout  and  cultures  made  from  the  pipettes  after  the 
termination  of  the  experiment.  That  contamination  was  infre- 
quent may  be  due  in  part  to  bactericidal  properties  of  the  emulsion 
or  of  the  methylene  blue,  or  of  both.  Experiments  which  showed 
contamination  were  discarded.  Reduction  was  allowed  to  take 
place  at  room  temperature. 

There  is  a  quantitative  relationship  in  the  reduction  of  methyl- 
ene blue  by  nervous  tissue.  In  one  case  were  used  0.5  c.c.  of 
emulsion  and  varying  quantities  of  methylene  blue  (o .  i  drop 
to  16  drops  of  a  methylene  blue  solution  which  contained  5  c.c. 
of  a  saturated  alcoholic  solution  of  the  dye  and  95  c.c.  sterile  NaCl 
solution).  One- tenth  to  one-half  of  a  drop  was  completely  re- 
duced; in  tubes  with  0.5-5  drops  partial  reduction  occurred,  the 
color  in  succeeding  tubes  increasing  gradually.  In  the  remaining 
tubes  the  overlying  fluid  was  densely  blue,  while  the  sediment  and 
a  very  small  zone  above  were  decolorized  after  twenty-four  hours. 
The  fact  that  such  an  emulsion  will  reduce  methylene  blue  at  all 
probably  indicates  that  intact  cells  are  not  essential.  An  emul- 
sion kept  in  the  ice-chest  for  a  week  will  reduce,  although  less 
vigorously  than  when  fresh.  It  occurred  to  Neisser  and  Wechs- 
berg  that  this  might  be  true,  for  they  say:  "It  is  entirely  con- 
ceivable that  certain  cells  may  contain  substances  which  in 
themselves  have  a  reducing  power,  without  regard  to  whether 
the  cells  as  such  are  living  or  not." 

The  reduction  is  in  no  case  permanent.  Tubes  after  being 
completely  reduced  for  three  days  regain  the  original  color  when 
shaken  with  air. 

A  drop  of  formalin  added  to  0.5  c.c.  of  the  emulsion  inhibited 
reduction,  so  that  at  the  end  of  three  days  there  was  only  a  trace, 
the  control  reducing  completely  in  two  hours. 


Reduction  of  Methylene  Blue  by  Nervous  Tissue     247 

Exposing  the  emulsion  of  nervous  tissue  to  a  temperature  of 
70°  C.  for  thirty  minutes  destroys  its  reducing  power  (Table  i). 
Tubes  of  the  emulsion  were  treated  as  indicated  in  the  table; 
then  I  drop  of  the  methylene  blue  solution  was  added. 

TABLE  I. 
Effect  of  Heat  on  the  Reducing  Power. 

So°  C.  for  30  tain No  retardation;  complete  in  2  hrs. 

56°  C.  for  30  min Retarded;  not  complete  in  10  hrs. 

60°  C.  for  30  min Retarded;  not  complete  in  10  hrs. 

65°  C.  for  30  min Retarded;  partial  reduction  in  18  hrs.; 

J  complete  in  48  his. 

70-95°  C.  for  30  min No  reduction  in  24  hrs. 

Other  experiments  show  that  the  emulsion  also  loses  its  reducing 
power  when  heated  to  98°  C.  for  five  to  ten  minutes. 

Subjecting  the  nervous  tissue  to  the  action  of  fresh  (or  acti- 
vated) neurotoxic  serum  did  not  destroy,  or  even  decrease,  the 
reducing  power  of  the  emulsion.  In  Table  2  the  mixtures  were 
sealed  at  once  without  allowing  time  for  a  preUminary  digestive  (  ?) 
action  of  the  serum  upon  the  nervous  tissue. 

TABLE  2. 
The  Influence  of  NEtTROxoxic  Serum  on  the  Reducing  Po\ver  of  Nervous 
Tissue.    Serum    Obtained    from    Goose    after    Immunization    with 
Nervous  Tissue  of  Guinea-Pig. 


Emulsion 

Serum 

Meth.  Blue 
Solution 

o.s  c.c. 
O.S  c.c. 
o.s  c.c. 
o.s  c.c. 
o.s  c.c. 
o.s  c.c. 
0 

o.s  c.c. 
0.4  c.c. 
0.3  c.c. 
0.2  c.c. 
0.1  c.c. 

0 
o.s  c.c. 

S  drops 

Complete  reduction  in  i8  hrs. 
No  reduction 

Similar  results  were  obtained  when  fresh  rabbit  serum  was 
used  in  order  to  add  complement,  previous  experiments  having 
indicated  that  rabbit  serum  contains  complement  for  the  neurotoxic 
amboceptor  of  the  goose  serum. 

Other  experiments  showed  that  the  addition  of  neurotoxic 
serum  to  the  nervous-tissue  emulsion  intensified  the  reducing 
power  of  the  latter,  rather  than  inhibiting  or  destroying  it.  A 
tube  containing  0.5  c.c.  of  serum  reduced  much  more  quickly  than 
one  containing  0.05  c.c.     The  serum  alone  is  non-reducing. 

Similar  results  were  obtained  by  treating  the  nervous  tissue 


248  Contributions  to  Medical  Science 

with  an  immune  rabbit  serum.  Digestion  of  the  tissue  for  six 
hours  with  such  a  serum  resulted  in  an  increase  in  the  reducing 
power,  provided  the  serum  was  not  removed  by  washing.  If, 
however,  the  serum  had  been  removed  in  this  manner,  the  reducing 
power  was  decreased.     For  example: 

a)  Five  c.c.  of  an  emulsion  two  days  old  was  centrifugated  and  the  overlying 
fluid  drawn  off.  One  and  a  half  c.c.  of  an  active  neurotoxin  was  then  added  to  the 
sediment  and  the  mi.xture  kept  at  37°  C.  for  two  hours,  after  which  it  was  diluted  to 
the  original  volume  with  salt  solution. 

Test  I. — One  c.c.  of  a)  +  i  drop  methylene  blue  solution:  complete  reduction  in 
twelve  to  twenty-four  hours. 

Test  2. — a)  was  again  washed,  resulting  in  the  removal  of  a  large  part  of  the  serum : 
no  reduction  in  three  days. 

b)  Same  as  a),  except  that  normal  rabbit  serum  was  substituted  for  the  neurotoxin. 
Test  I. — Complete  reduction. 

Test  2. — No  reduction  in  three  days. 

c)  Same  as  a)  and  b),  except  0.85  per  cent  sodium  chloride  solution  was  substi- 
tuted for  serum. 

Test  I. — Partial  reduction. 

Test  2. — No  reduction  in  three  days. 

d)  Controls: 

1.  One  c.c.  of  untreated  emulsion  reduced. 

2.  Two  c.c.  of  pure  rabbit  serum  gave  no  reduction. 

It  seems,  then,  that  normal  serum  as  well  as  the  immune  serum 
intensifies  the  reducing  power.  It  is  also  evident  that  repeated 
washing  of  the  emulsion — c),  i  and  2 — eliminates  its  reducing 
power,  the  presence  of  which  in  the  original  emulsion  is  indicated 
by  Control  i.  The  conditions  suggest  that  in  the  reduction  of 
methylene  blue  by  the  nervous  tissue  and  serum,  as  well  as  by  the 
fresh  emulsion  without  serum,  there  may  be  a  co-operation  of 
two  substances,  neither  of  which  alone  is  reducing — c),  2  and 
Control  2. 

Inasmuch  as  repeated  washing  of  an  emulsion  inactivated  it, 
it  seemed  probable  that  separation  of  the  two  suspected  sub- 
stances might  be  accomplished  by  extraction.  Attempts  at  sepa- 
ration by  centrifugating  and  washing  a  perfectly  fresh  emulsion 
were  not  successful ;  but  after  it  had  been  allowed  to  extract  for 
one  to  several  days,  one  of  the  substances  passed  into  the  liquid 
portion  in  a  state  of  solution  or  suspension,  the  other  remaining 
incorporated  with  the  solid  tissue. 


Reduction  of  Methylene  Blue  by  Nervous  Tissue      249 


In  Table  3  the  emulsion  had  been  kept  in  the  ice-chest  for 
eight  days. 

TABLE  3- 
Separation  of  the  Reddcing  Substances  in  Nervods  Tissue  by  Extraction  and  Washing. 


24  hrs. 

48  hrs. 

72  hrs. 

I.  Unwashed  emulsion 

Partial  reduc. 

0 

0 

Reduced 

0 

Reduced 

0 
0 

0 

2.  Sediment  washed  three  times 

0 

4.  Washed  sediment +3  drops  of  guinea-pig  senun 

*  In  the  course  of  several  days  pure  serum  causes  partial  reduction  of  the  dye. 

It  appears,  then,  that  the  serum  of  guinea-pig  is  able  to  replace 
the  substance  which  was  extracted  from  the  tissue.  Normal 
rabbit  serum  answers  equally  well  for  reactivation. 

The  extract'  when  added  to  washed  sediment  caused  reduc- 
tion, although  either  alone  did  not  reduce.  Reduction  is  not  so 
vigorous,  however,  as  when  the  original  emulsion  is  used,  probably 
because  a  portion  of  one  or  both  substances  is  wasted  in  the  inter- 
mediate washings.  Filtering  the  extract  through  a  Berkefeld 
filter  removes  the  reactivating  substance  from  the  fluid.  This 
would  seem  to  indicate  either  that  the  substance  is  in  large  molecu- 
lar form,  or  that  it  is  associated  with  tissue  particles  in  sus- 
pension which  are  held  back  by  the  filter. 

The  effect  of  successive  washings  (which  means  the  progressive 
removal  of  the  soluble  substance)  is  seen  in  Table  4.  Extraction 
was  allowed  to  proceed  for  twenty-four  hours  in  the  refrigerator. 

TABLE  4- 
The  Effect  of  Repeated  Washings  on  the  Reducing  Power  of  the  Emxtlsion. 


Unwashed  emulsion . 


Washed  once — no  serum 

Washed  once — serum,  2  drops. 


Washed  twice — no  serum 

Washed  twice — serum,  2  drops. 


Washed  3  times — no  serum 

Washed  3  times — serum,  2  drop>s. 

Two  drops  of  serum  alone 


12  hours 


Complete 

o 
Complete 

o 
Partial  reduction 

o 
Partial  reduction 


24  hours 


Complete 

Trace  reduction 
Complete 

Slight  trace 
Complete 


Almost  complete 


72  hours 


Complete 

Trace  reduction 
Complete 

Trace  reduction 
Complete 


Complete 


It  is  seen  that  as  washing  progresses  there  is  a  slight  tardiness 
in  the  reactivation  of  the  sediment  by  serum. 

"  By  the  "extract"  is  meant  the  fluid  of  a  one  to  several  days  old  emulsion  after  the  sediment  has 
been  removed  by  centrifugation. 


2^0 


Contributions  to  Medical  Science 


A  fresh  emulsion  may  be  inactivated  by  heating  to  98°  for 
five  to  ten  minutes,  or  to  70°  for  thirty  minutes.  The  soluble 
substance  is  hereby  destroyed,  the  one  resident  in  the  tissue  sedi- 
ment still  being  capable  of  reactivation  by  serum.  The  rapidity 
and  completeness  of  reduction  are  proportionate  to  the  quantity 
of  serum  used  for  reactivation.  In  Table  5  the  emulsion  was  three 
and  one-half  days  old,  and  for  the  experiment  was  heated  to  98° 
for  ten  minutes  in  order  to  inactivate  its  reducing  power.  Guinea- 
pig  serum  four  days  old  was  used  to  reactivate.  The  unheated 
emulsion  reduced  completely  in  twenty-four  hours.  Three  drops 
of  the  serum  alone  did  not  reduce. 

TABLE  5- 

The  Ability  of  Normal  Serum  of  Guinea-Pig  to  Reactivate  the  Reducing  Power.  After  the 

Latter  Had  Been  Destroyed  by  Heat. 


Emulsion 

Serum 

6  hours 

24  hours 

48  hours 

72  hours 

I.O  c.c 

0 

0 

0 

0 

0 

I.OC.C 

3  drops 

Almost  complete 

Complete 

Complete 

Complete 

I.OC.C 

2  drops 

Partial 

'* 

" 

1.0  c.c 

I  drop 

Trace 

Almost  complete 

'* 

" 

1.0  c.c 

i  drop 

0 

Marked  reduction 

" 

** 

I.O  c.c 

i  drop 

0 

0 

Partial 

Marked 

I.OC.C 

i^a  drop 

0 

0 

Trace  (?) 

Trace 

After  five  to  eight  days  both  substances  may  exist  to  some 
extent  in  the  extract,  since  it  is  possible  to  render  it  reducing  by 
the  addition  of  serum. 

In  order  to  determine  whether  or  not  the  reactivating  power 
of  serum  depends  on  complement,  guinea-pig  serum  was  heated 
to  different  temperatures,  and  its  reactivating  power  then  tested 
(Table  6).  In  this  experiment  a  5  per  cent  emulsion  of  nervous 
tissue  was  extracted  for  two  days,  then  inactivated  by  three 
washings.     The  serum  was  from  freshly  drawn  blood. 

In  this  experiment  it  is  seen  that  the  heating  of  the  serum, 
rather  than  destroying  the  reactivating  power,  has  increased  it, 
in  which  respect  there  is  no  resemblance  to  ordinary  complement. 
It  is  also  different  from  the  "soluble"  substance  of  the  extract  as 
a  reactivator,  since  the  latter  is  destroyed  by  a  temperature  of 
70°  for  thirty  minutes.  A  difference  from  ordinary  complement  is 
also  shown  by  the  fact  that  an  old  serum  will  serve  to  reactivate. 
Temperatures  higher  than  70°  were  not  tried. 


Reduction  of  Methylene  Blue  by  Nervous  Tissue     251 


TABLE  6. 

The  Effect  of  Heat  upon  the  Reacting  Power  of  Normal  Serum  of  Guinea-Pig. 

Washed  sediment  (i  .o  c.c.  in  each  tube)  to  which  was  added  i,  2,  and  3  drops  of  serum,  the  latter 
having  been  heated  to  the  different  temperatures  for  thirty  minutes. 

The  washed  sediment  in  each  case  was  suspended  in  the  original  volume  of  0.85  per  cent  NaCl. 


Temp. 

Observation 
Time 

Serum,  i  Drop 

Serum,  2  Drop>s 

Serum,  3  Drops 

40" 

12  hours 
20      " 
33      " 

0 
Almost  complete 
Complete 

0 
Complete 

o(?) 
Complete 

45° 

12      " 
20      " 
3i      " 

0 
Almost  complete 
Complete 

0 
Cornplete 

Slight  trace 
Complete 

so" 

la     " 
20     " 
33      " 

0 
Complete 

Trace 
Complete 

Trace 
Coniplete 

5S* 

12      " 
20     " 
33      " 

0 
Complete 

Partial 
Complete 

Almost  complete 
Complete 

6o' 

12      " 
20      " 
33      " 

0 
Partial  (I) 
Partial  (!) 

Marked 
Complete 

Almost  complete 
Complete 

6S* 

12       " 

20     " 
33      " 

Trace 
Complete 

Almost  complete 
Complete 

I 

70* 

12      " 

20      " 

33      " 

Almost  complete 
Complete 

Almost  complete 
Complete 

" 

Controls. — (i)  Unaltered  emulsion:    complete  reduction  in  twelve  hours 

(2)  Extract:  no  reduction  in  twenty-four  hours. 

(3)  Washed  sediment:  no  reduction  in  thirty-sLx  hours. 

(4)  Washed  sediment -|-fresh,  unhealed  guinea-pig  serum: 


I  Drop 


2  Drops 


3  Drops 


Twelve  hours 

Twenty  hours 

Thirty-three  hours . 


Partial 
Complete 


Trace 
Complete 


Marked 
Complete 


(s)  3  drops  of  secum  heated  to  40-70°  C.  for  thirty  minutes  showed  no  reduction  in  thirty-three 
I. 


hours. 


Influence  of  acids  and  alkalies. — Hydrochloric  acid,  except 
when  an  exceedingly  minute  amount  is  added,  destroys  the  redu- 
cing powers  of  the  fresh  emulsion.  It  will  not  reactivate  an 
inactive  sediment.  Quantities  of  o.  i-o.  2  c.c.  of  normal  potassium 
hydrate  solution  will  reactivate  the  boiled  emulsion  or  the  sedi- 
ment which  has  been  made  inactive  by  washing.  Smaller  quan- 
tities of  the  alkali  have  a  noticeable,  but  weaker,  effect.  Potassium 
hydrate  alone  does  not  reduce  methylene  blue,  although  it  affects  the 
dye  chemically,  as  shown  by  the  appearance  of  a  lavender  color. 


252  Contributions  to  Medical  Science 

Influence  of  tetanus  toxin. — Tetanus  toxin  in  any  quantity 
does  not  affect  the  reducing  power  of  nervous  tissue  for  methylene 
blue. 

SUMMARY  AND   CONCLUSIONS. 

The  ''bioscopic"  method  proved  unsuitable  as  a  means  of 
determining  the  toxic  action  of  neurotoxic  serum,  owing  possibly 
to  the  fact  that  this  serum,  in  common  with  normal  serums,  con- 
tains properties  adjuvant  to  the  reduction  of  methylene  blue. 

In  the  reduction  of  methylene  blue  by  nervous  tissue  the  living 
cell  is  not  essential. 

Reduction  by  the  tissue  emulsion  is  accomplished  by  the  com- 
bined action  of  two  substances,  one  of  which,  a  thermolabile 
substance,  may  be  extracted  from  the  tissue  by  a  0.85  per  cent 
NaCl  solution;  the  other,  a  thermostabile  substance,  is  closely 
associated  with  the  solid  tissue. 

Serum,  whether  it  is  old,  fresh,  or  heated  to  70°  for  thirty 
minutes,  may  be  substituted  for  the  thermolabile  substance,  as 
also  may  potassium  hydrate. 

Chemists  have  determined  that  the  reduction  of  methylene 
blue  may  be  accomplished  only  by  the  action  of  nascent  hydrogen 
on  the  dye.  It  becomes  leuko-methylene-blue  when  its  affinities 
for  hydrogen  have  been  satisfied.' 

In  the  original  emulsion  this  hydrogen  may  have  its  source  in 
fermentation  processes  (glycolytic  or  proteolytic),  since  certain 
ferments  and  the  substances  which  they  specifically  affect  are 
known  to  exist  normally  in  tissues  and  body  fluids. 

When  serum  is  used  as  a  reactivator,  it  is  questionable  if  the 
reduction  can  be  referred  to  the  action  of  ordinary  ferments 
in  view  of  the  heat  resistance  of  the  substance  contained  in  the 
serum  (70°  for  thirty  minutes).  The  serum  may  contain,  in 
addition  to  ferments,  some  obscure  catalyzing  agent  which  acts 
chemically  upon  substances  contained  in  the  tissue. 

It  would  seem  that  the  action  of  potassium  hydrate  must  be 
referred  to  its  catalyzing  properties. 

'  Boiling  a  solution  of  glucose  and  sodium  carbonate  which  contains  methylene  blue,  or  the  hydrogen 
evolved  by  the  action  of  a  mineral  acid  on  tin  in  a  methylene  blue  solution,  accomplishes  the  reduction 
of  the  dye.    Potassium  ferricyanide  reduces,  the  ferrocyanide  oxidizes,  the  dye. 


Reduction  of  Methylene  Blue  by  Nervous  Tissue     253 

It  has  not  been  possible  to  determine  the  amboceptor  and 
complement  nature  of  the  reaction  under  discussion,  since  the 
binding  phenomenon  could  not  be  demonstrated;  however,  a 
degree  of  analogy  is  not  lacking. 

A  few  experiments  performed  with  5  per  cent  emulsions  of 
liver  and  kidney  tissues  indicate  that  similar  principles  prevail  in 
the  reduction  of  methylene  blue  by  these  tissues. 

Although  a  most  interesting  chemical  problem  is  concerned  in  the 
phenomenon  presented,  one  is  confronted  with  so  many  unknown 
substances  and  reactions  that  the  writer  has  not  thought  it  profit- 
able at  present  to  follow  this  work  further.  It  is  not  unhkely  that 
methylene  blue  may  be  made  to  play  the  part  of  an  indicator  of 
reduction  reactions  where  sterile  substances  can  be  dealt  with. 


CONCERNING    THE    POSSIBILITY    OF    AN    ANTIBODY 
FOR  THE  TETANOPHILE  RECEPTOR  OF  ERY- 
THROCYTES:   A  RECEPTOR  STUDY.' 

H.     T.     RiCKETTS. 
{Prom  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

In  the  Transactions  of  the  Chicago  Pathological  Society,  1904, 
6,  p.  237,^  I  gave  a  brief  summary  of  some  experiments,  the  object 
of  which  was  to  determine  the  abihty  or  inability  of  various  recep- 
tors to  cause  the  formation  of  specific  antibodies.  We  are  accus- 
tomed to  say  that  those  receptors  of  erythrocytes  with  which  the 
hemolytic  amboceptors  unite  in  the  process  of  hemolysis  or  in  the 
binding  experiment  are  the  particular  constituents  of  the  cor- 
puscles which  cause  the  formation  of  the  amboceptors  when  the 
cells  are  injected  into  the  body  of  an  animal.  The  amboceptors 
are  in  this  sense  antibodies  for  the  corresponding  receptors  of  the 
erythrocytes.  Similarly,  the  receptors  of  corpuscles  with  which 
agglutinin  unites,  and  those  of  bacteria  with  which  the  bactericidal 
amboceptors  and  the  agglutinating  bodies  unite,  have  as  their 
specific  antibodies  the  hemagglutinin,  the  bactericidal  ambocep- 
tors, and  the  bacterium-agglutinin  respectively. 

The  question  arises:  Is  this  a  general  law?  Are  all  bodies 
which  function  as  receptors  capable  of  giving  rise  to  the  forma- 
tion of  specific  antibodies  when  they  are  injected  into  a  foreign 
species  ? 

The  question  has  appeared  to  the  writer  to  be  one  of  funda- 
mental importance,  especially  as  it  would  seem  to  have  a  direct 
bearing  on  the  ability  of  certain  pathogenic  bacteria  to  give  rise 
to  the  formation  of  antitoxins.  The  body  of  the  tj'phoid  bacillus 
contains  a  toxic  substance  which  we  reasonably  suppose  to  be  the 
pathogenic  constituent  of  the  organism.  Yet  immunization  with 
this  toxin-containing  bacterium  does  not  cause  the  formation  of 
an  indisputable  typhoid  antitoxin.     One  may  assume,  on  the  one 

^  Jour.  Exp.  Med.,  1905,  7,  p.  351., 

■"Receptor  Studies  Suggested  by  the  Side-Chain  Theory  of  Immunity";  see  also  "The  Effect  of 
Tetanolysin  on  Sensitized  Erythrocytes,"  Trans.  Chic.  Path.  Soc,  1905,  6,  p.  288. 

254 


Antibody  for  Tetanophile  Receptor  of  Erythrocytes   255 

hand,  that  the  specific  toxin  substance  of  the  typhoid  bacillus  is 
a  true  toxin,  with  a  haptophorous  and  a  toxophorous  group,  but 
that  the  conditions  in  the  body  are  such  that  a  specific  antitoxin 
cannot  be  formed.  The  same  argument  naturally  would  apply 
to  several  other  pathogenic  organisms.  Food-stuffs  and  various 
constituents  of  cells  are  present  in  the  body  continuously,  which, 
according  to  the  theory  of  Ehrlich,  may  be  taken  up  by  the  recep- 
tors of  the  cells,  but  for  which  it  is  thought  antibodies  do  not 
develop  {horror  autotoxicus) .  The  particular  receptors  with  which 
these  substances  unite  may  not  be  over-produced.  It  is  with 
receptors  of  this  nature  that  a  typhoid  toxin,  for  example,  may 
imite  and  yet  not  stimulate  to  the  formation  of  typhoid  anti- 
toxin. On  the  other  hand,  it  is  possible  that  the  essential  patho- 
genic substance  of  the  bacillus  is  not  a  toxin  in  the  sense  of  Ehrlich. 

It  is  the  purpose  in  this  paper  to  present  the  results  obtained 
in  an  attempt  to  demonstrate  the  presence  or  absence,  in  an  immune 
hemolytic  serum,  of  an  antibody  capable  of  uniting  with  those 
receptors  of  erythrocytes  for  which  tetanolysin  has  an  affinity. 
We  may  speak  of  these  receptors  as  the  tetanophile  receptors  of 
the  erythrocytes;  of  those  with  which  the  hemolytic  amboceptors 
unite  as  the  serum-lysophile  receptors,  and  of  those  with  which 
serum  agglutinin  unites  as  the  serum- agglutinophile  receptors. 

It  must  be  frankly  stated  at  the  outset  that  the  results  obtained 
throw  no  light  on  the  question  which  was  proposed,  yet  it  is  thought 
that  the  conditions  chosen  for  the  experiments  and  the  difficulties 
which  have  been  encountered  should  be  published. 

If  an  antibody  for  the  tetanophile  receptor  is  formed  when 
red  blood  corpuscles  are  injected  into  an  animal,  the  process, 
according  to  the  theory  of  Ehrlich,  would  be  the  following:  When 
the  erythrocytes  are  injected  they  disintegrate,  are  dissolved  or 
digested,  the  receptors  being  thus  hberated.  The  tetanophile 
molecules  or  receptors  would  eventually  reach  a  group  of  cells 
which  contain  molecules  or  receptors  with  which  the  former  could 
unite.  In  accordance  with  the  general  law  the  latter  would  then 
be  stimulated  to  over-production  and  many  of  them  thrown  into 
the  general  circulation.  When  the  serum  of  the  immunized 
animal  is  collected  and  mixed  with  erythrocytes  of  the  type  injected, 


256  Contributions  to  Medical  Science 

the  antibodies  for  the  tetanophile  receptors  should  unite  with  the 
latter  as  they  exist  in  the  red  corpuscles.  What  might  follow 
this  union  would  depend  on  the  character  of  the  antibody.  Inas- 
much as  hemolysis  and  agglutination  are  the  only  reactions  observ- 
able when  corpuscles  and  serums  are  mixed,  it  could  not  be  stated 
definitely  that  an  antibody  for  the  tetanophile  receptor  would 
not  be  able  to  cause  solution  of  the  corpuscles  or  their  agglutina- 
tion. In  this  case  the  antibody  would  be  a  receptor  of  the  second 
or  third  order,  since  it  would  be  possessed  of  a  toxophorous  con- 
stituent. If,  however,  it  were  a  receptor  of  the  first  order  it  would 
be  passive  in  its  nature,  since  such  receptors  possess  no  ferment- 
like or  toxic  action. 

For  the  purpose  of  the  experiments  it  was  assumed  that  an 
antibody  for  the  tetanophile  receptor  would  unite  with  the  latter 
and  thus  make  impossible  the  anchoring  of  tetanolysin,  just  as 
inactive  agglutinoids  unite  with  the  agglutinophile  receptors  of 
bacteria  and  stand  in  the  way  of  subsequent  union  of  the  latter 
with  active  agglutinin.  Under  the  assumption,  the  antibody 
in  question  and  the  tetanolysin  molecule  would  have  identical 
haptophore  groups.  It  is  appreciated  that  even  if  the  supposed 
antibody  is  formed,  difficulties  might  stand  in  the  way  of  its 
demonstration  by  the  method  chosen.  In  the  first  place,  the 
heating  of  the  serum,  which  was  found  to  be  necessary  in  order  to 
destroy  its  hemolytic  power,  might  destroy  the  antibody.  Again, 
special  temperature  conditions,  other  than  those  utilized,  might 
be  necessary  for  union.  Or  union  having  occurred,  it  might  be 
an  easily  dissociable  one;  the  tetanolysin  might  have  a  greater 
affinity  for  the  tetanophile  receptor  than  the  antibody  has,  and  on 
this  account  be  able  to  displace  the  latter,  or  the  union  might  be 
dissociated  by  subsequent  washing  with  salt  solution. 

The  method  of  experimentation  consisted  of  exposing  a  definite 
quantity  of  washed  erythrocytes  to  the  action  of  varying  quantities 
of  the  heated  immune  hemolytic  serum  for  different  lengths  of 
time  and  at  different  temperatures,  after  which  the  serum  was 
partially  or  totally  removed  by  centrifugation,  and  the  effect  of 
tetanolysin  then  observed  on  the  corpuscles.  The  heating  of  the 
immune  serum  was  found  to  be  necessary  inasmuch  as  I  did  not 


Antibody  for  Tetanophile  Receptor  of  Erythrocytes   257 


succeed  in  preventing  some  subsequent  hemolysis  in  the  binding 
experiment  with  unheated  serum  at  low  temperatures.  When 
such  hemolysis  occurred  it  naturally  rendered  an  estimation  of  the 
action  of  the  tetanolysin  impossible.  Normal  serums  used  as  con- 
trols were  treated  in  the  same  way. 

Inasmuch  as  the  tetanophile  and  the  serum-lysophile  receptors 
are  not  identical,  it  was  assumed  that  the  absorption  of  the  ambocep- 
tors did  not  prevent  subsequent  fixation  of  the  tetanolysin  by  the 
tetanophile  receptors.  If  the  two  receptors  were  identical,  anti- 
tetanolysin  should  have  a  haptophore  capable  of  uniting  with  the 
cytophilous  haptophore  of  the  amboceptor.  Experiments  showed 
that  heated  normal  goat  serum,  rich  in  antitetanolysin,  when 
added  to  fresh  immune  hemolytic  serum  from  the  goat,  did  not 
reduce  the  dissolving  power  of  the  latter  serum.  For  a  similar 
reason  it  was  concluded  that  the  tetanophile  receptor  is  not  identi- 
cal with  the  agglutinophile  receptor. 

TABLE  I. 
The  Effect  of  Saponin  on  Corpuscles  Which  Were  Agglutinated  by  Ricin. 
Ricia  and  saponin,  each,  o.  2  per  cent  solution;  5  per  cent  emulsion  of  washed  guinea-pig  erythrocytes 
in  0.85  per  cent*  salt  solution;  corpuscles  and  ricin  at  37°  for  thirty  minutes;  ricin  removed  by  repeated 
centrifugation;  o.i  c.c.  saponin  solution  added. 


Ricin 

Agglutination 

Saponin 

Hemolysis 

i.o    c.c. 

Complete 

0. 1  c.c. 

Trace 

0-5 

0.3 

0.1 

0.0s 

0.03 

O.OI 

Partial 

" 

Slight 

Moderate 

Marked 

Complete 

0 

0 

•  Throughout  the  experiments  o .  85  per  cent  salt  solution  and  i  c.c.  of  a  5  per  cent  emulsion  of  erythro- 
cytes were  used.    Total  volume  in  all  tubes  was  2  c.c. 

The  first  important  complication  which  developed  consisted 
of  agglutination  of  the  corpuscles  by  the  serum.  Although  the 
agglutinated  mass  could  be  broken  up  into  very  fine  particles  by 
pipetting  the  solution  back  and  forth,  nevertheless  such  corpuscles 
often  sank  to  the  bottom  readily  and  became  reagglutinated. 
The  mere  sedimentation  of  the  corpuscles  renders  them  physically 
less  accessible  to  the  action  of  the  tetanolysin.  It  is  possible 
also  that  the  alteration  of  the  envelope  of  the  corpuscle  which 
seems   to   occur   in   agglutination   renders   them   less   susceptible 


258  Contributions  to  Medical  Science 

to  the  action  of  a  hemolytic  agent.  Corpuscles  (guinea-pig) 
which  have  been  agglutinated  by  ricin  or  abrin  are  to  a  large  degree 
protected  from  the  hemolytic  action  of  saponin,  although  they  are 
readily  hemolyzed  when  distilled  water  is  added.  The  smaller 
the  amount  of  ricin  used  the  greater  the  hemolysis  which  occurs 
when  saponin,  not  a  true  toxin,  is  added  subsequently. 

Here  also  there  may  be  chemical  or  physical  changes  in  the 
envelope,  or  the  union  of  hemoglobin  with  the  stroma  may  be 
made  more  firm  by  the  action  of  the  ricin,  but  the  physical  pro- 
tection of  agglutination  cannot  be  disregarded. 

Guinea-pig  erythrocytes  which  are  treated  with  a  heated 
immune  hemolytic  serum  (rabbit)  are  also  made  resistant  to  the 
action  of  saponin,  the  corpuscles  having  been  strongly  agglutinated 
by  the  serum. 

Experiments  with  Saponin  and  Tetanolysin. 

1.  Control:  corpuscles  were  completely  hemolyzed  by  o.i  c.c.  saponin  solution 
in  a  few  minutes. 

2.  Erythrocytes  were  treated  with  0.3  c.c.  heated  immune  serum  for  thirty 
minutes;  washed  free  of  serum;  o.i  c.c.  saponin  solution:  no  hemolysis.  These 
corpuscles  may  subsequently  be  hemolyzed  by  distilled  water. 

3.  Corpuscles  without  saponin  or  serum  show  no  hemolysis. 

4.  Non-agglutinating  normal  serum  gave  no  protection. 

The  following  is  an  analogous  experiment  performed  with  the  heated  immune 
serum  (rabbit),  and  tetanolysin: 

1.  Control:  0.6  c.c.  of  0.2%  tetanolysin  solution  hemolyzed  the  corpuscles 
of  guinea-pig  completely  in  thirty  minutes. 

2.  Corpuscles  without  tetanolysin  or  serum  show  no  hemolysis. 

3.  Corpuscles  treated  with  0.3  c.c.  serum  for  thirty  minutes;  serum  removed 
by  washing;  0.6  c.c.  tetanolysin  solution:  no  hemolysis  in  twenty-four  hours.  The 
corpuscles  were  shaken  and  the  clot  broken  up  repeatedly  during  this  time.  These 
corpuscles  were  subsequently  hemolyzed  by  distilled  water. 

4.  Non-agglutinating  normal  serum  afforded  no  protection. 

In  view  of  the  evident  influence  of  agglutination  as  a  protective 
condition  against  the  action  of  hemolyzing  substances,  the  pro- 
tection obtained  in  the  experiment  last  cited  cannot  logically  be 
assigned  to  a  preoccupation  of  the  tetanophile  receptors. 

More  recently  experiments  have  been  resumed  using  a  heated 
hemolytic  serum  obtained  from  the  goat  by  immunization  with 
the  erythrocytes  of  the  sheep.  A  serum  rich  in  hemolytic  ambo- 
ceptors, but  poor  in  agglutinins,  is  obtained,  as  pointed  out  by 


Antibody  for  Tetanophile  Receptor  of  Erythrocytes   259 

Ehrlich  and  Morgenroth.  It  was  thought  that  agglutination 
as  a  complicating  factor  might  be  eliminated  by  the  use  of  this 
serum.     This  proved  not  to  be  the  case. 

Apparent  protection  was  obtained  in  many  experiments,  even 
when  the  agglutination  caused  by  the  serum  was  slight,  as  shown 
in  Table  2.  The  serum  (goat  i)  and  blood,  thoroughly  mixed 
and  diluted  uniformly  to  2  c.c,  were  placed  at  37°  for  thirty  minutes. 
Tubes  were  centrifugated,  and  the  overlying  fluid  drawn  of!, 
leaving  a  total  residue  of  about  one-twentieth  of  a  cubic  centimeter. 
The  tetanolysin  (i .  o  c.c.  of  a  o .  2  per  cent  solution)  was  then  added, 
each  tube  diluted  to  2  c.c.  and  the  corpuscles  thoroughly  mixed 
by  pipetting  back  and  forth.  That  the  agglutinated  corpuscles 
were  largely  separated  in  this  way  is  shown  by  microscopic  exami- 
nation, in  which  no  more  than  three  to  six  corpuscles  were  grouped 
together.     Tubes  were  examined  after  twenty-four  hours. 

The  following  experiment  seems  to  lose  significance  when  a 
duplicate,  in  which  the  serum  was  still  further  removed  by  an 
additional  washing,  showed  that  the  immune  serum  had  no,  or 
little,  more  protective  action  than  the  normal  serums.  Yet 
inasmuch  as  in  this  experiment  two  normal  serums  were  used  as 
controls,  and  the  latter  showed  a  lower  protection  than  the  immune 
serum,  there  must  have  been  present  some  property  in  the  immune 
serum,  which  in  the  normal  serums  was  absent,  or  present  in  lower 
concentration. 

There  are  two  possibilities  by  way  of  explaining  the  loss  of 
protection  occasioned  by  the  washing,  aside  from  the  influence 
of  agglutination.  In  the  first  place,  an  antibody  for  the  tetano- 
lysin receptor  may  unite  with  the  latter  in  the  more  concentrated 
serum  mixture,  but  the  dilution  involved  in  the  washing  may 
cause  dissociation  of  the  union,  so  that  after  the  second  centrifuga- 
tion  the  tetanophile  receptors  may  again  be  available  for  union 
with  tetanolysin.  In  the  second  place,  some  other  protective 
factor  may  be  present  in  both  the  immune  and  normal  serums, 
and  in  a  particular  experiment  might  be  present  in  greater  concen- 
tration in  the  immune  serum  than  in  the  two  normal  serums. 
Antitetanolysin,  or  some  substance  of  equivalent  power,  as  choles- 
terin,  may  be  present  In  the  minute  residuum  of  serum  left  in  the 


26o 


Contributions  to  Medical  Science 


original  tubes,  and  it  might  in  a  given  instance  be  present  in  greater 
quantities  in  the  immune  serum  than  in  the  normal.  The  cor- 
puscles treated  with  the  immune  serum  would  in  this  instance  be 
protected  to  a  greater  extent  than  those  treated  with  the  normal 
serum.  If  the  serum  left  in  the  sediment  after  the  first  centrifuga- 
tion  is  0.008  c.c.  (estimated),  a  subsequent  washing  in  2  c.c.  of 
salt  solution  reduces  the  residual  serum  to  a  quantity  which  has 
no  antitoxic  effect  on  the  amount  of  tetanolysin  used,  as  shown 
by  experiments. 

TABLE  2. 

The  Action  oy  Tetanolysin  on  Sheep's  Erythrocytes  Which  Had  Been 

Treated  with  Heated  Immune  Hemolytic  SERim  from  the  Goat. 


Immune 
Serum 

Nonn.  Ser. 
No.  I 

Norm 
No 

Ser. 
2 

Tetano- 
lysin 

Hemolysis 

I 

2 

3 

0.8 

0.8 

0 

8 

I.O 

No  hemolysis 
SUght 

4 
S 
6 

0.6 

0^6 

0 

6 

I 

No  hemolysis 
Slight 

7 
8 

0.4 

0.4 

" 

Trace 
SUght 

9 

0 

4 

10 
II 
12 

0.2 

0.2 

0 

2 

I 

Trace 

Moderate 

Marked 

13 

14 

0.1 

0.1 

« 

Moderate 
Marked 

IS 

0 

I 

16 

17 

0 
0 

0 
0 

0 
0 

0 

Complete 
No  hemolysis 

Inasmuch  as  various  quantities  of  serum  were  used  in  the 
different  tubes,  a  higher  degree  of  protection,  which  the  larger 
amounts  of  serum  may  have  afforded,  might  be  referred  to  the 
larger  amount  of  antitoxin  present  in  those  tubes.  To  eliminate 
this  factor  the  amounts  of  residual  serum  in  the  various  tubes 
were  equalized  by  washing  with  proportionate  amounts  of  salt 
solution  (Table  3). 

Since  a  large  proportion  of  the  protection  in  A  was  removed 
by  the  washing,  there  must  be  present  a  soluble  antitoxic  substance 
in  the  serum. 

If  we  assume  that  the  inhibition  of  tetanolysin  hemolysis  in 
the  above  experiment  was  due  to  two  factors,  agglutination  and  an 


Antibody  for  Tetanophile  Receptor  of  Erythrocytes   261 


antitetanolytic  property  in  the  serum,  and  that  in  the  washing 
the  antitetanolysin  was  equaHzed,  we  have  the  following  values 
for  the  two  factors  in  those  tubes  in  which  0.6  c.c.  of  serum  was 
used: 

Total  inhibition  90%        80%        40%        30% 

Antitoxin  inhibition  70%         70%        40%         30% 

Agglutination  inhibition  20%         10%  o  o 

These  percentages  are  only  of  comparative  value,  inasmuch  as 
somewhat  more  than  the  simple  dissolving  dose  of  tetanolysin 
was  used,  and  at  the  same  time  the  antitetanolysin  in  the  different 
tubes  was  equalized  but  not  entirely  removed. 

TABLE  3. 
A. — Corpuscles  of  the  sheep  exposed  to  action  of  immune  serum  from  the  goat,  tubes  centrifugated, 
overlying  fluid  drawn  off;  corpuscles  not  washed;  tetanolysin  is  i  .0  c.c.  of  a  o.  2  per  cent  solution. 


Seruh 
Amount 


Subsequent  Hemolysis  by  Tetanolysin 


Immune  Ser. 
No.  1 


Immune  Ser. 
No.  2 


Normal  Ser. 
No.  I 


Normal  Ser. 
No.  2 


.6  c.c. 

■4 


10% 

10 

25 

7S 

Control:  100  per  cent 


20% 
30 
60 
100 


60% 

80 
100 
100 


70% 
100 
100 
100 


B. — Duplicate  of  the  above  except  that  the  quantities  of  residual  serum  in  the  different  tubes  were 
equalized  by  washing  with  proportionate  amounts  of  salt  solution. 


Serum 

Salt  Sol. 
Used  in 
Washing 

Subsequent  Hemolysis  by  Tetanolysin 

Immune  Ser. 
No.  I 

Immune  Ser. 
No.  2 

Normal  Ser. 
No.  r 

Normal  Ser. 
No.  2 

I 

3 

3 

4 
S 

0.6  c.c. 
0.4 
0.2 
0.1 
0 

2.4  c.c. 
1.6 
.8 

•  4 
0 

80% 

80 
100 
1 00 
Control :     i 

90  % 
80 
90 
100 
30  per  cent 

100  % 
1 00 
100 
100 

100% 
100 
100 
100 

The  corpuscles  treated  with  the  immune  serums  were  rather  firmly  agglutinated. 

Nothing  is  deducible  concerning  the  presence  or  absence  of 
an  antibody  for  the  tetanophile  receptor.  Any  possible  pro- 
tection afforded  by  the  presence  of  this  antibody  cannot  be  sepa- 
rated from  the  protection  due  to  the  agglutination. 

Something  might  be  learned  concerning  the  nature  of  the  pro- 
tection by  fractional  absorption  of  the  immune  bodies  from  the 
serum,  and  testing  the  action  of  tetanolysin  on  the  erythrocytes 
which  were  used  for  the  removal  of  the  successive  fractions  of 


262  Contributions  to  Medical  Science 

immune  bodies.  Quantities  of  0.6,  0.4,  0.2,  o.i  c.c.  of  the  dif- 
ferent serums  were  diluted  to  2  c.c.  each,  and  the  contents  of  each 
tube  added  to  the  sediment  of  i  c.c.  of  the  5  per  cent  blood  emulsion. 
After  thirty  minutes  at  room  temperature  the  tubes  were  centrifu- 
gated,  the  overlying  fluid  drawn  off  and  put  into  fresh  tubes, 
while  to  the  sediment  was  added  the  usual  amount  of  tetanolysin 
in  a  total  volume  of  2  c.c.  The  serum  dilutions  were  then  sub- 
jected to  the  absorbing  powers  of  a  second  mass  of  corpuscles  of 
equal  amount  and  the  process  repeated  over  four  absorptions, 
the  hemolytic  power  of  the  tetanolysin  being  tested  on  the  red 
blood  corpuscle  sediments  of  each  series.  At  the  same  time  a 
series  of  tubes  was  made  to  determine  the  relative  antitoxin  con- 
tent of  each  of  the  serums  (Table  4,  A  and  B). 

There  are  three  sources  of  error  which  may  be  responsible  for 
some  variations  in  the  percentages  of  hemolysis  in  the  different 
tubes  and  in  the  different  series  in  A.  First,. after  the  corpuscles 
were  exposed  to  the  action  of  the  serum  and  the  tubes  were  centrifu- 
gated,  the  overlying  serum  mixtures  were  drawn  off  as  completely 
as  possible  by  means  of  a  glass  pipette  which  was  drawn  to  a  fine 
point;  in  spite  of  care  the  amounts  of  residual  serum  in  the  different 
tubes  were  undoubtedly  subject  to  variations,  so  that  Tube  i, 
for  example,  in  the  column  headed  "Immune  Serum  No.  i," 
might  contain  in  the  different  series  variable  amounts  of  residual 
serum — that  is,  variable  amounts  of  antitoxin  along  with  other 
serum  constituents.  Second,  the  completeness  with  which  the 
agglutinated  corpuscles  in  the  tubes  treated  with  the  immune 
serums  were  broken  up,  probably  was  not  constant  in  spite  of 
caution.  Third,  the  experiment  occupied  a  whole  day  and  the 
tetanolysin  lost  a  small  percentage  of  its  strength  during  that  time, 
although  it  was  kept  on  ice  when  not  in  use. 

Analysis  of  the  columns  headed  "Normal  Serum,"  in  which 
agglutination  was  absent,  shows  that  although  a  moderate  loss 
in  the  protection  of  the  corpuscles  takes  place  as  absorption  con- 
tinues, the  degree  of  protection  corresponds  to  the  relative  amounts 
of  antitoxin  in  the  two  serums  as  shown  in  the  antitoxin  table. 
All,  or  nearly  all,  the  protection  may  reasonably  be  assigned  to  the 
antitoxin.     Inasmuch  as  the  total  volume  of  the  sediment  and 


Antibody  for  Tetanophile  Receptor  of  Erythrocytes   263 

TABLE  4- 

A. — The  effect  of  tetaaolysin  on  corpuscles  which  have  been  used  for  the  fractional  absorption  of 
antibodies  from  the  serums. 

First  Series. 


Immune  Serum  No.  i 

Immune  Serum  No.  2 

Normal  Ser.  No.  1 

Normal  Ser.  No.  2 

Agglut.  by 
Serum 

Hemol.  by 
Toxin 

Agglut.  by 
Serum 

Hemol.  by 
Toxin 

Agglut.  by 
Serum 

Hemol.    by 
Toxin 

Agglut.  by 
Serum 

Hemol.   by 
Toxin 

I 

3 

3 
4 

Firm 
Less 

Marked 

10 
10 

25 

75 

Very  firm 
Less 

Firm 

20 
30 
60 
1 00 

0 
0 
0 
0 

60 
80 
100 
too 

0 
0 
0 
0 

70 
100 
100 
100 

S 

No  serum, 

100  per  cent  hemolysis: — Control. 

Second  Series. 


Third  Series. 


I 

Gradually 

IS 

Gradually 

35 

0 

70 

0 

go 

2 

less  from 
I  to  4 

20 

decreased; 
more  than 

65 

0 

85 

0 

90 

3 

20 

by  Im- 

100 

0 

100 

0 

100 

4 

75 

mune  Se- 
rum No.  I 

100 

0 

100 

0 

100 

I 

Gradually 

10 

Gradually 

75 

0 

,0 

0 

80 

2 

less  from 

25 

less  from 

90 

0 

90 

0 

100 

^ 

I  to  3 

6S 

I  to  4 

100 

0 

100 

0 

100 

4 

No  agglut. 

80 

100 

0 

100 

0 

100 

Fourth  Series. 


I 

No  agglut. 

10 

Moderate 

95 

0 

70 

0 

80 

2 

No  agglut. 

15 

Agglut. 

95 

0 

0 

100 

3 

No  agglut. 

55 

Slight 

100 

0 

lOO 

0 

100 

4 

No  agglut. 

65 

100 

0 

100 

0 

100 

B. — Antitetanolysin  in  the  four  serums. 

One  cubic  centimeter  of  a  0.2  per  cent  solution  of  tetanolysin  is  mixed  with  the  quantities  of  the  serums 
stated  in  the  table,  in  a  total  volume  of  2.0  c.c.  After  two  hours'  contact  at  room  temperature  the  con- 
tents of  each  tube  is  added  to  the  red  corpuscle  sediment  of  i  .0  c.c.  of  the  5  per  cent  blood  suspension. 


Serum 

Immune  Ser. 

Immune  Ser. 

Normal  Ser. 

Normal  Ser. 

No.  I 

No.  2 

No.  I 

No.  2 

.005 

20 

50 

45 

60 

.0045 

20 

60 

55 

60 

.004 

25 

70 

70 

60 

.0035 

30 

70 

75 

65 

.003 

35 

75 

75 

70 

.0025 

45 

90 

90 

75 

.002 

6S 

100 

100 

90 

.0015 

8S 

100 

100 

90 

.001 

90 

100 

100 

95 

.0005 

90 

100 

100 

100 

0 

Control :    1 

00  per  cent 

264  Contributions  to  Medical  Science 

residual  fluid  in  each  tube  was  about  0.03  c.c,  the  antitoxin  was 
diluted  by  that  much  each  time  the  serum  mixture  was  added  to 
a  fresh  lot  of  corpuscles.  This,  however,  amounts  to  a  loss  of 
only  about  0.25  per  cent  from  the  first  to  the  third  series  and  is 
a  negligible  quantity.  The  corpuscles  also  may  have  absorbed 
some  of  the  antitoxin,  a  possibility  which  was  not  determined. 

Analysis  of  the  columns  headed  "Immune  Serum,"  in  which 
agglutination  was  present,  shows  a  great  loss  of  protection  between 
the  first  series  and  the  third,  and  the  loss  is  more  uniform  and 
greater  with  the  serum  which  had  the  higher  agglutinating  power; 
hand  in  hand  with  the  loss  of  protection  is  the  progressive  decrease 
in  the  agglutinating  power  of  the  serum  as  absorption  continues. 
The  greater  protection  with  Immune  Serum  No.  i ,  which  was  pres- 
ent in  spite  of  its  lower  agglutinating  power,  is  explained  when 
we  note  its  greater  antitoxic  power  (Table  4,  B). 

This  experiment,  then,  and  duplicates  which  have  been  made 
afford  no  clue  as  to  the  presence  of  an  antibody  for  the  tetano- 
phile  receptor;  the  protection  which  was  encountered  is  explain- 
able on  the  basis  either  of  agglutination  or  antitoxin  action. 

Other  experiments  were  performed  with  the  view  of  deter- 
mining more  exactly  the  antitoxic  value  of  the  residual  serum  in 
the  different  tubes.  The  results  showed  that  in  the  case  of  normal 
serums  the  antitoxin  of  the  residual  serum  accounted  for  practi- 
cally all  of  the  protection,  while  in  the  case  of  the  immune  serums 
in  which  some  agglutination  had  occurred,  the  calculated  amount 
of  antitoxin  did  not  account  for  all  the  protection.  The  difference 
in  protection  by  the  two  immune  serums  used  corresponded  to 
the  differences  in  the  agglutinating  and  antitoxic  powers  of  the 
two  serums.  Here  also  no  means  developed  of  distinguishing 
an  antibody  for  the  tetanophile  receptor  from  the  agglutinating 
and  antitoxic  powers  of  the  serums.  Attempts  to  eliminate 
agglutinin  by  heat  failed  as  the  serums  produced  some  aggluti- 
nation even  after  exposure  to  the  temperature  of  coagulation. 

SUMMARY  AND   CONCLUSIONS. 

The  treatment  of  washed  erythrocytes  of  the  guinea-pig  by 
heated  immune  hemolytic  serum  derived  from  the  rabbit,  and 


Antibody  for  Tetanophile  Receptor  of  Erythrocytes   265 

of  washed  erythrocytes  of  the  sheep  by  a  similar  serum  derived 
from  the  goat,  renders  the  erythrocytes  more  or  less  resistant  to 
the  subsequent  action  of  tetanolysin. 

If  all  the  serum  is  removed  from  corpuscles  treated  in  this 
manner,  it  can  be  determined  that  some  of  the  protection  is  due 
to  the  agglutination  of  the  cells.  A  certain  amount  of  the  protec- 
tion afforded  by  agglutination  is  referable  to  the  physical  barrier 
which  the  agglutinated  mass  of  cells  offers  to  the  uniform  distribu- 
tion and  diffusion  of  the  tetanolysin.  It  is  probable  that  none 
of  the  protection  obtained  is  due  to  the  mere  union  of  agglutinin 
or  of  hemolytic  amboceptors  with  their  respective  cell  receptors; 
such  union  would  appear  to  leave  the  tetanophile  receptors  still 
unoccupied. 

If  a  residuum  of  serum  is  left  with  the  corpuscles  which  have 
been  treated  as  indicated,  the  added  protection  which  is  acquired 
against  the  subsequent  action  of  tetanolysin  may  reasonably  be 
referred  to  antitoxin  which  is  present  in  the  residual  serum.  The 
possibility  of  the  dissociation  of  the  union  between  the  tetano- 
phile receptor  and  its  antibody,  occasioned  by  the  dilution  inci- 
dent to  washing  away  the  serum,  may  not  be  entirely  ignored, 
but  has  not  been  susceptible  to  determination.  The  same  may 
be  said  of  the  possibility  that  tetanolysin,  having  a  stronger  affinity 
for  the  tetanophile  receptor  than  has  the  antibody,  is  able  to 
displace  the  latter  from  its  union  with  the  receptor. 

Hence  treating  corpuscles  with  the  immune  serums  does  not 
allow  one  to  determine  the  presence  or  absence  of  an  antibody 
for  the  tetanophile  receptor,  the  experiments  being  formulated 
on  the  supposition  that  such  an  antibody  would  unite  with  the 
tetanophile  receptors  and  thereby  prevent  subsequent  binding 
of  tetanolysin. 


THE   ADJUVANT  ACTION   OF   SERUM,  EGG-ALBUMIN, 
AND  BROTH  ON  TETANUS  INTOXICATION.' 

H.     T.     RiCKETTS     AND     E.     J.     KiRK. 

(From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

In  the  course  of  other  work  it  became  necessary  to  assure  our- 
selves concerning  the  content  in  tetanus  antitoxin  of  the  sera  of 
several  goats.  To  our  surprise  normal  goat  serum  (i  .o  c.c),  rather 
than  exerting  an  antitoxic  action,  increased  the  toxicity  of  the 
toxin  when  suitable  doses  of  the  latter  were  used.  The  investiga- 
tion of  this  phenomenon  is  the  subject  of  the  present  paper. 

TECHNIC   AND  MATERIALS. 

The  toxin  used  throughout  the  experiments  was  prepared  in 
January,  1903,  as  follows:  Ten  liters  of  beef  broth,  containing  i 
per  cent  of  glucose  and  having  a  reaction  of  -f-i,  were  placed  in 
two  bottles  and  inoculated  with  a  large  quantity  of  tetanus  bacilli 
which  had  grown  for  10  days  in  a  similar  broth.  Washed  hydrogen 
was  then  passed  through  the  inoculated  broth  for  an  hour,  and  the 
bottles  sealed  and  placed  in  the  thermostat,  the  usual  provisions 
being  made  to  permit  of  the  escape  of  gas  from  the  bottles,  and  to 
prevent  the  access  of  air.  After  a  growth  of  nine  days  the  culture 
was  passed  through  Pukal  filters,  placed  in  large  moisture  dishes, 
and  an  excess  of  ammonium  sulphate  added  ;^  the  dishes  were 
then  placed  in  the  thermostat  over  night.  The  brownish  scum 
which  had  formed  by  this  time  was  skimmed  oflf,  placed  between 
hardened  filter  papers,  and  the  excess  of  moisture  pressed  out. 
Still  more  fluid  and  ammonium  sulphate  were  removed  by  sub- 
jecting the  precipitate  to  very  high  pressure  in  a  pressure  machine. 
The  precipitate,  now  in  the  form  of  solid  cakes,  was  dried  over 
sulphuric  acid  and  eventually  pulverized.  It  is  preserved  over 
sulphuric  acid  in  the  ice-chest  and  in  the  dark. 

For  use  a  o .  2  per  cent  solution  of  the  precipitate  was  made  in 
0.85  per  cent  sodium  chloride  solution,  and  the  doses  used  are 

■  Jour.  Infect.  Dis.,  1906,  3,  p.  116. 

'  One-half  more  than  the  quantity  the  broth  would  dissolve  at  room  temperature. 

266 


Adjxa'^ant  Action  of  Serum  on  Tetanus  Intoxication    267 

expressed  in  cubic  centimeters  of  this  solution.  The  original  fatal 
dose  for  white  mice  of  about  15  grams  weight  was  0.000,007  c.c. 
per  gram  of  mouse;  death  occurred  in  four  to  five  days.  For 
guinea-pigs  of  250  to  300  grams,  0.000,001  c.c.  per  gram  killed  in 
four  to  five  days.  The  toxin  has  undergone  little  or  no  deteriora- 
tion since  it  was  prepared. 

When  dealing  with  a  substance  of  such  high  toxicity  it  is  difii- 
cult  to  weigh  out  small  quantities  with  desirable  accuracy  and  at 
the  same  time  observe  economy  of  material.  Our  toxin  solutions 
were  usually  made  by  dissohdng  o .  2  gram  of  the  precipitate 
in  100  c.c.  of  the  salt  solution,  but,  in  spite  of  great  care  in  weighing, 
variations  in  the  toxicity  of  different  solutions  frequently  came  to 
light.  Some  of  these  variations  may  have  been  due  to  an  irregular 
distribution  of  the  toxin  in  the  precipitate.  A  new  toxin  solution 
was  prepared  for  each  experiment. 

White  mice  were  used  as  the  test  animals  throughout  the  work, 
and  the  dosage  of  toxin  was  based  on  the  gram-weight  of  the 
animals — i.e.,  so  much  toxin  per  gram.  It  was  found,  however, 
that  this  method  could  be  used  safely  only  when  the  various  mice 
of  a  given  experiment  were  approximately  of  the  same  weight, 
since,  for  example,  0.000,007  c.c.  of  toxin  per  gram  is  not  equally 
toxic  for  a  20  gram  and  a  10  gram  mouse.  Susceptibility  is  not 
directly  proportional  to  weight. 

The  injections  were  made  into  the  loose  subcutaneous  tissue  of 
the  back. 

We  soon  learned  that  the  ability  of  normal  serum  to  increase 
the  toxicity  of  the  toxin  disclosed  itself  only  when  suitable  doses 
of  the  toxin  were  used.  If  a  dose  of  toxin  which  killed  all  controls 
in  48  to  72  hours  was  used,  the  increased  toxicity  caused  by  the 
addition  of  serum  did  not  become  manifest.  Also  if  the  dose  was 
so  small  that  very  Uttle  tetanus  resulted  in  any  of  the  animals,  the 
effect  of  the  serum  was  not  always  a  decisive  one.  The  clearest 
results  were  obtained  when  a  dose  of  toxin  was  used  which  either 
caused  moderate  tetanus  in  the  controls  with  eventual  recovery, 
or  which  caused  their  death  in  from  eight  to  twelve  days. 

Because  of  the  possibility  of  variations  in  the  toxicity  of  toxin 
solutions,  one  could  not  feel  sure  that  a  single  dose  selected  for  an 


268  Contributions  to  Medical  Science 

experiment  would  be  the  optimum  dose  for  the  manifestation  of 
the  phenomenon.  Hence  it  was  decided  to  use  a  number  of  mice 
for  each  experiment,  and  to  vary  the  dosage  in  such  a  way  that 
an  optimum  dose  would  be  administered  to  two  or  more  of  the  ani- 
mals.    It  was  necessary  to  use  an  abundant  number  of  controls. 

The  sera  of  a  number  of  normal  goats  were  used,  also  of  normal 
rabbits,  and  for  specific  purposes  which  will  be  explained  later, 
the  influence  of  broth  and  of  egg-albumin  was  tested.  Cus- 
tomarily I  c.c.  of  the  serum  (or  broth,  or  egg-albumin)  was  mixed 
with  a  dilution  of  the  toxin  of  which  0.5  c.c.  contained  the  desired 
dose.  The  mixture  was  allowed  to  stand  for  varying  lengths  of 
time  and  the  total  quantity  (i .  5  c.c.)  then  injected  into  the  sub- 
cutaneous space  in  the  back  of  the  white  mouse  by  means  of  a 
Luer  or  Roux  syringe.  After  the  mixture  had  been  injected  an 
additional  0.5  c.c.  of  salt  solution  was  drawn  into  the  syringe, 
and,  after  rinsing,  was  injected.  In  order  to  prevent  any  escape 
of  fluid  after  its  injection,  the  needle  puncture  was  clamped  for 
15  to  30  minutes,  the  clamp  being  applied  before  the  needle  was 
withdrawn. 

In  all  experiments  daily  observations  were  made  of  the  degree 
of  tetanus  present,  as  indicated  by  the  amount  of  deformity  and 
the  general  appearance  of  the  animals.  The  degree  of  tetanus  is 
expressed  in  the  tables  by  figures:  thus  o  =  no  tetanus;  1=  per- 
ceptible rigidity;  2  =  distinct  but  not  pronounced;  3  =  marked 
rigidity;  4  =  severe;  5  =  very  severe  to  moribund;  t  =  death.  The 
observations  of  one  of  us  served  as  a  check  on  those  of  the  other. 


EXPERIMENTS. 

Tables  i,  2,  and  3,  which  follow,  are  typical  experiments  show- 
ing the  influence  of  normal  goat  and  rabbit  sera. 

Tables  1,2,  and  3  require  little  or  no  comment.  Particularly 
in  Tables  i  and  2,  there  can  be  no  question  concerning  the  ability 
of  the  serum  to  increase  the  intensity  of  the  tetanus  intoxication 
from  which  death  occurred.  Table  3  is  somewhat  less  decisive, 
but  here  also  all  animals  receiving  serum  died  from  one  to  three 
days  in  advance  of  the  controls. 


Adjuvant  Action  of  Serum  on  Tetanus  Intoxication    269 


TABLE  1. 
The  Intluence  of  Normal  Goat  Serum  ok  Tetanus  Intoxication. 
Normal  serum,  Goat  I. — Serum  three  days  old,  unhealed,  preserved  in  the  ice-chest.     Serum  and 
toxin  mixed  and  allowed  to  stand  30  minutes  at  37°  C.  before  injection. 


Mouse 

Wt.  in 

Grams 

Toxin 
PER  Gram 

C.C.  OF 

Serum 

Result  by  Days 

1 

2 

3 

4 

S 

6 

7      8 

9 

lO 

11 

IS 

1 

a 

14 
14 
16 
16 
14 
14 
14 
14 
13 
13 

0.000,001 

0.000,001 

0.000,003 

0 .  000,003 

0.000,006 

0.000,006 

0 .  000,008 

0 .  000,008 

0.000,01 

0.000,01 

1 .0 
0.0 
1 .0 
0.0 
1 .0 
0.0 
1 .0 
0.0 
1 .0 
0.0 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

I 
0 
0 

1 
2 

I 
I 
2 
3 

1 

2 
I 
2 
2 
3 
I 
4 
2 

5 
2 

I 
I 
2 
2 

4 

I 
t 
3 
t 
3 

I 
I 
2 

I 
4 

I 

3 
3 

1 
I 
2 

1 
4 

I 

3 
3 

I 
3 

I 

I 
I 
0 

I 

0 
3 

I 

I 
0 
I 
0 

0 

3 

I 

I 
0 
I 
0 

0 

2 

I 

I 
0 
I 
0 

0 

I 
0 

0 
0 

4 

5 

6 

7 

0 

8 

9 

0 

TABLE  2. 
Same  as  Table  i,  except  that  the  serum  of  Goat  II  was  used. 


Mouse 

Wt.  in 
Grams 

Toxin 
PER  Gram 

C.C.  OF 

Serum 

Resttlt  by  Days 

I 

2 

3 

4 

S 

6 

7 

8 

9 

10 

II 

12 

IS 

I 

14 
14 
15 
15 
14 
14 
.14 
14 
14 
14 

0.000,001 

0.000,001 

0.000,003 

0 .  000,003 

0 .  000,006 

0.000,006 

0.000,008 

0 .  000,008 

0.000,01 

0.000,01 

1.0 
0.0 
1.0 
0.0 
1.0 
0.0 
1 .0 
0.0 
1 .0 
0.0 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

I 
0 
2 

I 
I 
1 
2 
I 
3 
2 

I 
I 
3 
I 
3 
3 
3 
3 
5 
2 

1 
I 
4 
I 
3 
3 
S 

3 

I 
I 
4 
2 
4 
2 
t 
4 

I 
I 
4 
3 
4 
2 

S 
1 

1 
1 
4 
3 
4 
I 

t 
2 

I 
I 
S 
3 
4 
1 

2 

0 
0 

t 

2 
4 

I 

2 

0 
0 

2 

t 
2 

0 
0 

2 

2 

0 
0 

I 

I 

0 
0 

0 

|:;:::::;:;:;: 

0 

Gost) 

TABLE  3- 
The  Influence  of  Normal  Rabbit  Serum  on  Tetanus  Intoxication. 
Serum,  unheated,  two  days  old.     Mixtures  stood  for  one  and  one-half  hours  at  room  temperature 
before  injection. 


Mouse 

Wt.  IN 
Grams 

Toxin  per 
Gram 

C.C.  OF 

Serum 

Result  by  Days 

1 

2 

3 

4 

S 

6 

7 

I 

2 

3 

13 
11 
16 
IS 
19 
16 

0.000,003 
0.000,003 
0.000,006 
0 .  000,006 
0 .  000,008 
0 .  000,008 

1.0 
0.0 
1 .0 
0.0 
1 .0 
0.0 

0 
0 
0 
0 
0 
0 

2 
2 
3 
3 
4 
2 

3 
3 

4 
4 
t 
2 

3 

5 

4 

S 
4 

S 

t 

t 
S 

t 

t 

6:::::::::::::::::: 

In  accordance  with  Table  4,  o.  i  c.c.  of  serum  is  a  more  power- 
ful adjuvant  than  i  c.c,  the  latter  being  the  dose  used  uniformly. 
This  point  was  determined  only  after  the  major  part  of  the  experi- 
ments had  been  completed.     Its  explanation  is  by  no  means  clear. 


270 


Contributions  to  Medical  Science 


The  serum  may  contain  a  minute  amount  of  antitoxin,  which 
declares  itself  when  larger  quantities  are  used. 

TABLE  4. 
To  Determine  the  Minimum  Qoantity  of  Goat  Serum  Which  Intensifies  the  Intoxication. 
Fresh  serum,  18  hovirs  old,  from  a  normal  Angora  goat. 


Mouse 
Wt.  10  Grams 

Toxin  per 
Gram 

C.C.  OF 

Serum 

Result 

BY  Days 

I 

2 

3 

4 

S 

6 

0 .  000,008 
0.000,008 
0.000,008 
0 .  000,008 
0 .  000,008 
0.000,008 
0 .  000,008 
0.000,008 

0.0 
0.0 
0.01 
0.0s 

O.IO 

0.40 
0.70 
1. 00 

0 
0 
0 
0 
0 
0 
0 
0 

0 
2 

I 
2 
2 

I 
I 
I 

2 
3 
3 

3 

I 
I 

4 
4 
t 
t 

S 
3 
3 

S 
S 

t 
5 
t 

t 

t 

6::::::::::::::;::::::::::::::::: 

7 

8     

t 

Experiments,  of  which  Table  5  is  an  example,  were  performed 
to  determine  whether  the  efifect  of  the  serum  could  be  attributed  to 
its  toxic  action  on  the  mice.  If  such  a  toxic  action  were  present, 
the  early  death  of  the  animals  might  be  referred  to  a  summation 
of  the  intoxication  by  the  serum  and  that  by  the  toxin.  We 
looked  on  the  subsequent  weight  of  the  animals  as  a  clue  to  the 
presence  or  absence  of  serum  intoxication. 

TABLE  5. 
To  Determine  the  Toxicity  or  Non-Toxicity  of  Normal  Goat  Serum  for  White  Mice. 
Serum  three  days  old,  unheated. 


Mouse 

Wt.  IN 
Grams 

C.C.    OF 

Serum 

Weight  by  Days 

I 

2 

4 

S 

6 

17 
14 
17 

I.O 

i-S 
2.0 

18 
IS 
18 

I9S 
17.0 
20.0 

18 
16 
20 

18 
IS 
18 

18 

2 

IS 
18 

There  was  no  loss  of  weight,  but  even  a  gain  on  the  second  day, 
which  may  have  been  due  to  the  drinking  of  more  water.  Hence 
we  conclude  that  the  phenomenon  cannot  be  explained  by  any 
manifest  toxicity  of  the  serum  for  the  mice. 

Other  experiments  were  performed  with  the  hope  that  some 
Hght  might  be  thrown  on  the  nature  of  the  phenomenon.  It 
occurred  to  us  that  tetanus  toxin  might  be  an  amboceptor,  and 
that  the  normal  serum  of  the  goat  might  contain  suitable  com- 


Adjuvant  Action  of  Serum  on  Tetanus  Intoxication   271 

plement  which,  when  injected,  would  increase  the  amount  of 
suitable  complement  in  the  body  of  the  mouse,  and  thus  activate 
the  toxin  more  quickly  and  more  completely.  To  throw  possible 
light  on  this  point  the  comparative  influence  of  fresh,  old,  and 
heated  sera  was  determined  (Table  6). 


TABLE  6. 
The  Comparative  Influence  of  Fresh,  Heated,  and  Old  Sera  on  Tetanus  Intoxication. 
The  sera  were  from  Goat  IV  (female);   the  "heated"  serum  had  been  placed  at  56°  C.  for  30  min- 
utes and  was  freshly  drawn;   the  "old"  serum  had  been  drawn  11  days  previously  and  kept  at  about 
10°  C. 


Mouse 

Wt.  in 
Grams 

Toxin  per 
Gram 

I  c.c. 

OF 

Serum 

Result  by  Days 

I 

2 

3 

4  !   S 

6 

7 

8 

1 
9    |IO 

II 

14 

19 

I 

2 

18 
18 
18 
18 
18 
18 
18 
18 

0.000,006 
0.000,006 
0.000.006 
0.000,006 
0.000,008 
0.000,008 
0 .  000,008 
0.000,008 

Fresh 
Heated 
Old 
None 
Fresh 
Heated 
Old 
None 

0 
0 
0 
0 
0 
0 
0 
0 

I 
I 
I 

I 
0 

I 
I 
I 

2 
2 
2 
2 
3 
3 
3 
2 

3 
3 
3 
3 
3 
4 
t 
3 

4 
3 

1 

3 

4 
3 
t 
3 
4 

3 

>' 

S 
S 

4 
4 

t 
t 

3 
3 

2 
2 

3 

I 

3 

5 

6 

7 

8 

The  experiment  shows  distinctly  that  the  effect  of  the  serum 
does  not  depend  on  an  activation  of  hypothetical  tetanus  ambo- 
ceptors by  ordinary  thermolabile  complement.  The  heated  and 
old  sera  were  rather  more  efficient  than  the  fresh  serum.  Serum 
heated  even  to  the  coagulating  point  does  not  lose  its  adjuvant 
property  (Table  7). 

TABLE  7. 
Heat  Resistance  of  the  Adjuvant  Substance. 


Mouse 
Wt.  12  Grams 


Toxin  per 
Gram 


I  c.c.  Goat 

Serum  Heated 

TO  — °C 

FOR   30   MIN. 


Result  by  Days 


3 
4 
S 
6 
7 
8 
9 


10 


000,007 
.  000,007 
.000,007 
.000,007 
.000,007 
.000,007 
.000,007 
.  000,007 
.000,007 
.  000,007 


67.5-68* 
67.S-68 
64 . 5-65 
64  5-65 
59 ■ 5-60 
59 ■ 5-60 
Unheated 
Unhealed 
No  serum 
No  serum 


•  The'serum  at  67 .  s°-68''  was  gelatinous.    Temperatures  higher  than  68°  C.  were  not  tried. 

Other  experiments  seem  to  show  that  the  influence  of  the  serum 
does  not  depend  on  any  action  on  the  toxin  itself.     It  is  not  neces- 


272 


Contributions  to  Medical  Science 


sary  to  mix  the  serum  and  toxin  before  injection  in  order  to  get 
the  effect  of  the  former.  The  two  may  be  injected  in  different 
parts  of  the  body,  or  the  serum  may  be  injected  shortly  in  advance 
of  the  toxin.  It  is  well  known,  on  the  other  hand,  that  a  substance 
which  acts  directly  on  the  toxin  (tetanus  antitoxin)  has  a  stronger 
effect  when  the  two  are  mixed  before  injection.  In  order  to  get 
the  maximum  effect  of  the  serum,  however,  it  is  necessary  that  the 
two  substances  be  injected  in  fairly  close  sequence  although  they 
may  be  placed  in  different  parts  of  the  body  (Table  8). 

TABLE  8. 
The  Effect  of  the  Serum  When  Injected  at  Different  Periods  in  Advance  of  the  Toxm. 


I  CO.  Serum 
Hours  in 

Mice 

10 
Grams 
Weight 

Toxin  per 
Gram 

Result  by  Days 

Advance  of 
Toxin 

I 

2 

3 

4 

S 

6 

7 

8 

9 

10 

It 

12 

0  1  2 
0     3 

0  1  3 
0  1  3 

2 
4 
3 
4 
4 
2 

3 

4 
4 

4 
t 
3 

4 
4 
S 
4 

3 

4 
S 
t 
S 

4 

3 

5 

t 
4 

3 
5 

4 

4 
t 

S 

t 

21      "     

"    2 

"     3 
"     4 
"     5 
"     6 

0 .  000,008 
0.000,008 
0  000,008 
0 .  000,008 
0.000,008 

No  serum 

0 

2 

t 

The  results  of  this  experiment  give  us  the  impression  that  the 
action  of  the  serum  depends  on  some  temporary  influence  which  it 
exerts  on  the  tissues  of  the  animal,  whereby  the  latter  is  made 
more  susceptible  to  the  toxin.  After  the  injection  of  the  serum 
its  influence  gradually  becomes  less,  and  is  not  demonstrable 
after  the  lapse  of  about  46  hours. 

It  is  equally  important  from  the  standpoint  of  interpretation  to 
know  whether  the  serum  intensifies  intoxication  when  given  sub- 
sequent to  the  injection  of  the  toxin.  The  result  of  a  single  experi- 
ment indicates  that  the  serum  does  not  hasten  the  death  of  the 
animals  when  it  is  given  subsequent  to  the  binding  of  the  toxin  by 
the  tissues.  The  controls  which  received  serum  and  toxin  simulta- 
neously died  in  five  days,  while  animals  in  which  the  injection  of 
the  serum  was  given  from  one  to  fifty  hours  later  than  that  of  the 
toxin  either  recovered  or  died  in  from  six  to  nine  days. 

We  had  come  to  believe  at  this  time  that  there  was  nothing  of  a 
specific  nature  in  the  phenomenon,  and  that  many  other  proteid- 
containing  substances  might  have  a  similar  influence.     We  found 


Adjuvant  Action  of  Serum  on  Tetanus  Intoxication    273 


this  to  be  strikingly  true  in  the  cases  of  egg-albumin  and  broth 
(Tables  9  and  10). 

We  have  before  us  then  the  following  facts  upon  which  we  may 
attempt  to  base  conclusions: 

1 .  The  normal  sera  of  the  goat  and  rabbit  intensify  and  hasten 
the  course  of  tetanus  in  white  mice,  when  suitable  doses  of  our 
precipitated  toxin  are  used  (Tables  1,2,  and  3). 

2.  It  is  immaterial  whether  the  serum  is  fresh,  old,  or  heated 
to  the  coagulating  point  (Tables  6  and  7). 

3.  The  effect  is  most  pronounced  when  the  serum  is  injected 

TABLE  9. 

The  Influence  of  Egg-Albumin  on  Tetanus  Intoxication. 

One  per  cent  of  egg-albumin  in  0.85  per  cent  NaCl  solution. 


Result  by 

Days 

Mouse 

WT.  IN 

Grams 

Toxin  pee 
Gram 

C.C.  OF 

Albumin 

I 

2 

3 

4 

5 

6 

7 

8 

9 

I 

IS 

0.000,00s 

1       o.s 

0 

I 

2 

3 

4 

t 

2 

15 

0 .  000,005 

1       0.0 

0 

0 

0 

0 

I 

I 

0 

0 

0 

3 

IS 

0.000,006 

1       O-S 

0 

I 

2 

S 

t 

4 

IS 

o.ooo,co6 

0.0 

0 

0 

0 

I 

I 

I 

I 

I 

0 

s 

IS 

0.000,007 

o.s 

0 

I 

3 

t 

6 

IS 

0.000,007 

0.0 

0 

0 

I 

I 

I 

I 

I 

0 

0 

TABLE  10. 
The  Influence  of  Broth  on  Tetanus  Intoxication. 


Mouse 


Wt.  in 
Grams 


Toxin  per 
Gram 


C.C.   OF 

Broth 


Result  by  Days 


10    II,  12,  13 


a IS 

3 18 

4 18 

S '  IS 

6 ,  IS 


0.000,006 
0.000,006 
0.000,007 
0.000,007 
0.000,008 
0.000,008 


Recovery 


simultaneously  with,  or  shortly  preceding,  the  injection  of  the 
toxin.  If  the  serum  is  given  46  hours  in  advance  of  the  toxin  it  is 
in  some  way  disposed  of  so  that  it  no  longer  intensifies  the  intoxica- 
tion. It  is  not  necessary  that  the  serum  be  mixed  with  the  toxin, 
nor  injected  into  the  same  part  of  the  body  (Table  8). 

4.  A  small  quantity  of  serum  (o.i  c.c.)  seems  to  have  a  more 
pronounced  influence  than  a  larger  quantity  (i  c.c.)  (Table  6). 

5.  Normal  goat  serum  in  a  quantity  of   2  c.c.  produces  no 
perceptible  deleterious  effect  on  the  mouse  (Table  5). 


274  Contributions  to  Medical  Science 

6.  One-half  c.c.  of  a  i  per  cent  solution  of  egg-albumin  in 
physiologic  salt  solution;  and  also  i  c.c.  of  broth  have  an  influence 
like  that  of  serum. 

The  possibility  which  we  at  one  time  took  under  consideration 
that  tetanus  toxin  might  be  an  amboceptor,  and  that  the  serum 
increases  toxicity  because  it  provides  an  additional  quantity  of 
complement  cannot  be  entertained:  first,  because  old  and  heated 
sera,  in  which  there  is  no  complement  (thermolabile),  produce 
the  phenomenon;  and,  second,  because  egg-albumin  or  broth  may 
be  substituted  for  the  serum  with  the  same  result.  We  cannot 
accuse  the  broth,  in  particular,  of  containing  complement. 

Inasmuch  as  we  have  not  been  able  to  refer  the  phenomenon 
to  any  influence  which  the  adjuvant  substances  may  exert  on  the 
toxin,  we  have  been  obliged  to  assume  that  it  is  due  to  some  efifect 
on  the  tissues  of  the  mice.  Although  the  serum  exerted  no  per- 
ceptible toxic  action  on  the  mice,  which  if  it  had  occurred  might 
have  lessened  resistance  in  some  manner,  we  were  bound  to  con- 
sider as  a  possibility  that  the  serum,  albumin,  and  broth  might 
exert  some  particular  influence  on  the  nervous  tissue  whereby  it 
either  absorbed  more  toxin  or  was  made  less  resistant  to  the  toxin 
which  it  bound.  In  spite  of  this  possibility,  however,  we  have  been 
unable  to  conceive  of  any  manner  in  which  these  substances  could 
produce  such  an  effect  on  the  nervous  tissue.  If  the  serum,  etc., 
were  to  some  extent  bound  as  indifferent  food  substances  by  the 
nervous  cells,  we  believe  the  latter  might  be  preoccupied,  so  to  say, 
in  digesting  them;  but  since  such  a  process  would  engage  the  cells 
in  a  general  way  we  believe  their  affinity  for  some  second  sub- 
stance, such  as  tetanus  toxin,  would  be  decreased  rather  than 
increased  during  this  period.  Concerning  the  alternative  pos- 
sibility that  the  serum,  etc.,  may  injure  the  nervous  cells  so  that 
they  are  less  resistant  to  the  toxin,  we  have  only  the  argument  that 
no  injury  detectable  by  gross  means  was  produced. 

We  find  what  appears  as  a  more  reasonable  explanation  of  the 
phenomenon  in  an  influence  which  the  serum,  etc.,  may  exert  on 
the  remaining  tissues  of  the  body  other  than  the  nervous  tissue. 
We  learn  from  certain  investigations  by  Metschnikoff,'  and  by 

■  L'lmmuniti,  Paris,  1901,  p.  343. 


Adjuvant  Action  of  Serum  on  Tetanus  Intoxication   275 

Roux  and  Borrel/  that  other  tissues  than  the  nervous  are  able  to 
bind  tetanus  toxin  in  some  instances.  The  rabbit  and  also  the 
chicken  are  much  more  susceptible  to  tetanus  toxin  when  it  is 
injected  into  the  nervous  tissue  than  into  the  subcutaneous  tissue; 
in  the  latter  instance  the  toxin  comes  in  contact  with  various 
tissues  some  of  which  bind  a  certain  amount.  Metschnikoff  found 
that  the  liver  in  some  of  the  invertebrates  absorbs  a  great  deal  of 
tetanus  toxin.  We  are  not  aware  of  any  experiments  bearing  on 
the  antitoxic  powers  of  the  organs  of  the  white  mouse,  nor  have 
we  performed  such  experiments.  Wassermann  and  Takaki  found 
that  small  quantities  of  the  Hver,  kidney,  spleen,  and  bone-marrow 
of  the  guinea-pig  exerted  no  antitoxic  action,  a  condition  which 
corresponds  well  with  the  exquisite  susceptibility  of  this  animal  to 
tetanus.  In  comparing  the  susceptibility  of  the  rabbit  with  that 
of  the  guinea-pig,  we  learn,  first,  that,  gram  for  gram,  the  rabbit 
is  about  a  thousand  times  more  resistant  than  the  guinea-pig 
(Knorr,  cited  by  Dieudonne);^  and  second,  from  Roux  and  Borrel, 
that  a  large  part  of  this  resistance  resides  in  other  than  nervous 
tissues.  The  tetanus  sine  tetano  of  Donitz  suggested  to  him  that 
non-nervous  organs  in  the  rabbit  bind  tetanus  toxin. ^  Such 
animals  after  receiving  a  dose  of  toxin  which  causes  little  or  no 
tetanus  gradually  become  emaciated  and  die. 

We  cannot  of  course  hold,  without  definite  experimental  proof, 
that  the  same  conditions  exist  in  the  mouse.  We  know,  however, 
that  the  mouse,  gram  for  gram,  is  from  six  to  ten  times  more 
resistant  to  tetanus  toxin  than  the  guinea-pig  (Knorr);''  in  our 
own  experiments  it  is  seven  times  more  resistant,  and  it  is  quite 
possible  that  some  of  this  resistance  depends  on  the  abihty  of  tissues 
other  than  the  nervous  to  bind  a  certain  amount  of  the  toxin. 

If  this  condition  exists,  and  if  in  some  way  union  of  the  toxin 
with  other  tissues  could  be  prevented,  for  example,  with  the  liver, 
connective  tissues,  or  lymphoid  organs,  just  so  much  more  toxin 
would  be  available  for  the  more  susceptible  nervous  tissue. 

We  conceive  that  such  a  result  may  be  caused  by  the  serum, 

■  Ann.  de  I' Inst.  Pasteur,  1898,  12,  p.  229. 

'  Immunildt,  Schutzimp/ung,  etc.,  Leipzig,  1903,  p.  13. 

J  Deutsche  med.  Wchnschr.,  1897,  23,  p.  428. 

<  Cited  by  Dieudonn^,  loc.  cil. 


276  Contributions  to  Medical  Science 

etc.,  in  one  of  two  ways:  First,  certain  receptors  or  substances  in 
the  serum,  egg-albumin,  and  broth  may  possess  cytophilous 
haptophores,  identical  with  that  of  tetanus  toxin,  and  by  uniting 
with  the  tetanophile  receptors  of  indiflferent  organs  (liver,  etc.) 
may  thereby  render  impossible  the  binding  of  the  toxin;  this 
extra  toxin  would  then  be  available  for  the  nervous  tissue,  which 
presumably  has  a  higher  affinity  than  other  tissues  for  the  toxin. 
Such  a  process  would  consist  of  a  preoccupation  of  tetanophile 
receptors  by  heterologous  substances. 

Second,  the  cells  of  indiflferent  organs  (liver,  etc.)  may  bind  the 
serum,  etc.,  as  they  would  bind  food  substances,  after  which  their 
activities  (ferments)  may  be  directed  toward  the  digestion  or 
oxidation  of  the  new  substances,  and  being  thus  engaged,  the 
affinities  of  their  receptors  for  other  substances  (toxin)  may  be 
decreased.  This  condition  would  also  render  more  toxin  available 
for  the  nervous  tissue.  Such  a  process  would  consist  of  a  non- 
specific engagement  of  the  activities  of  the  cells,  without  a  direct 
occupation  of  tetanophile  receptors. 

As  an  example  of  such  a  process  we  may  mention  the  well- 
known  experiments  of  Besredka,'  in  which  granules  of  carmin 
injected  into  the  peritoneal  cavity  decreased  the  ability  of  the 
leukocytes  to  take  up  granules  of  arsenic,  with  the  result  that  the 
animals  died  the  more  readily  of  arsenic  intoxication. 

Von  Dungern  also  observed  a  clear  example  of  the  ability  of 
one  substance  to  interfere  with  the  absorption  of  a  second.^  By 
injecting  the  plasm  of  Octopus  vulgaris  or  egg-albumin  into  the 
circulation  of  a  rabbit  the  ability  of  the  animal  to  absorb  a  sub- 
sequent injection  of  the  plasm  of  Majo  squinado  (spider  crab)  was 
largely  or  entirely  inhibited.  Von  Dungern's  explanation  of  this 
phenomenon  is  somewhat  incomplete,  but  we  understood  it  to 
be  the  following:  In  reference  to  the  particular  substances  injected, 
the  cells  of  the  rabbit  have  two  types  of  receptors.  One  type  is 
that  which  is  able  to  take  up  various  materials  as  food  substances 
and  is  not  a  specific  receptor.  It  takes  up  egg-albumin,  the  plasm 
of  the  spider  crab  and  of  the  octopus,  and  doubtless  many  other 

•  Ann.  de  t'Insl.  Pasteur,  iSgg,  12,  pp.  49  and  209. 
'  Die  Antikorper,  Jena,  1903,  p.  97. 


Adjuvant  Action  of  Serum  on  Tetanus  Intoxication   277 

albumins.  It  is  not  concerned  in  the  formation  of  antibodies. 
The  other  type  is  specific,  can  take  up  only  one  particular  sub- 
stance, and,  when  it  proliferates,  is  cast  into  the  circulation  as  an 
antibody. 

Now  when  the  plasm  of  the  octopus  or  egg-albumin  was  injected 
into  the  rabbit,  the  first  type  of  receptor,  the  non-specific  type,  was 
occupied  largely  or  completely,  and  hence  was  not  able  to  fix  the 
plasm  of  the  crab  which  was  injected  two  and  one-half  hours 
later.  Only  the  second  type,  the  specific  receptor,  remained  to 
fix  the  plasm  of  the  crab.  As  a  consequence  the  last-named 
substance  disappeared  from  the  circulation  much  more  slowly 
than  when  it  was  injected  into  an  untreated  rabbit. 

We  interpret  the  phenomenon  which  we  have  described  in  a 
somewhat  similar  way,  although  it  is  necessary  for  us  to  introduce 
the  consideration  that,  the  tissues  of  the  mouse  having  united  with 
the  serum,  egg-albumin,  or  broth,  they  thereby  lose  in  their  afl&nity 
for  tetanus  toxin.  After  the  foreign  substances  have  been  dis- 
posed of  by  the  cells,  the  latter  again  reach  a  state  of  equilibrium, 
and  their  tetanophile  receptors  regain  their  former  affinity  for  the 
toxin  (Table  8). 

It  is  not  possible  to  assume,  on  the  basis  of  our  experiments, 
that  an  antitoxic  or  bactericidal  serum  can  in  any  way  intensify 
the  corresponding  disease,  provided  the  serum  is  sufficiently  rich  in 
antibodies.  In  diphtheria,  for  example,  the  quantity  of  antitoxin 
introduced  is  able  to  neutrahze  all  the  toxin  which  happens  to  be 
in  the  body,  hence  there  is  no  possibility  of  intensifying  the  diph- 
theritic intoxication. 


THE  STUDY  OF  "ROCKY  MOUNTAIN  SPOTTED  FEVER" 

(TICK  FEVER  ?)  BY  MEANS  OF  ANIMAL 

INOCULATIONS.^ 

A  PRELIMINARY  COMMUNICATION. 

H.     T.     RiCKETTS. 

I  arrived  in  Missoula,  Montana,  April  21,  1906,  equipped  for 
the  bacteriologic  and  hematologic  study  of  the  so-called  Rocky 
Mountain  spotted  fever  and  for  the  study  of  the  infectious  agent 
by  means  of  animal  inoculations. 

Although  the  period  during  which  the  disease  occurs  is  very 
limited  and  the  cases  very  few,  it  was  possible  to  carry  the  investiga- 
tions to  a  point  which  affords  encouragement  for  the  ultimate 
solution  of  some  ot  the  problems  involved.  At  this  time  I  wish 
to  give  a  brief  and  preliminary  presentation  of  the  most  important 
results  obtained  this  year,  reserving  for  a  future  date  a  more 
detailed  interpretation  of  the  experiments,  when  the  bacteriologic 
and  histologic  studies  will  have  been  completed. 

It  will  be  remembered  that  the  confidence  which  was  at  first 
manifested  in  the  report  of  Wilson  and  Chowning  concerning  the 
presence  of  a  piroplasma  in  the  blood  and  erythrocytes  of  infected 
patients  was  greatly  shaken  by  the  observations  of  Stiles,  who, 
after  prolonged  study,  was  utterly  unable  to  confirm  the  piroplas- 
matic  nature  of  the  disease. 

From  these  conflicting  reports  it  seemed  very  probable  that  the 
specific  organism,  if  present  in  the  blood,  must  be  one  which  is 
difficult  of  determination,  and  for  the  demonstration  of  which 
special  experimental  methods  must  be  used. 

The  first  essential  point  to  be  studied  concerned  the  situation 
of  the  specific  agent  in  the  body.  Considering  the  subject  from 
an  unprejudiced  point  of  view,  it  was  necessary  to  recognize  the 
possibility  ot  the  microbe  having  a  local  situation,  the  general 
disturbance  being  caused  by  toxins  which  are  absorbed  from  the 
point  of  primary  infection.     This  possibility  was  considered  some- 

'  This  work  was  done  in  part  under  a  grant  made  by  the  American  Medical  Association  through  the 
Committee  on  Scientific  Research.    From  Jour.  Am.  Med.  Assn.,  1906,  47,  p.  a. 

278 


The  Study  of  "Rocky  Mountain  Spotted  Fever"    279 

what  improbable,  however,  the  characteristic  and  generalized 
eruption  speaking  for  a  systemic  rather  than  a  local  infection. 
On  this  account,  my  attention  was  directed  chiefly  to  the  blood 
from  the  start. 

After  a  fairly  exhaustive  search  of  the  blood  in  stained  and 
unstained  preparations  in  two  cases,  I  was  unable  to  identify 
a  parasite  positively  either  within  or  without  the  erythrocytes. 
Similarly,  aerobic  and  anaerobic  cultures  of  the  blood,  made  in 
bouillon  and  on  agar  plates  of  differing  reactions,  gave  negative 
results  with  one  exception.  In  the  one  exception  mentioned, 
a  staphylococcus  grew  in  a  flask  of  bouillon  which  had  been  inocu- 
lated with  from  3  to  5  c.c.  of  blood  drawn  from  the  patient,  Landon. 

In  view  of  these  negative  results,  it  was  decided  to  proceed  at 
once  with  animal  inoculations,  although  the  microscopic  examina- 
tion of  the  blood  and  culture  experiments  were  continued  as  new 
cases  developed. 

experiments  on  rabbits. 

On  April  27,  two  intravenous  inoculations  were  made  into 
rabbits.  In  one  instance,  i  c.c.  of  defibrinated  blood  from  the 
patient,  Stevenson,  was  used,  and  in  the  other  4  c.c.  of  defibrinated 
blood  from  the  patient,  Landon.  The  Stevenson  blood  was  17 
hours  old  and  the  Landon  blood  24  hours  old  at  the  time  of  injec- 
tion, the  blood  having  been  kept  in  the  ice-chest  except  for  the 
first  few  hours  occupied  in  transporting  it  to  the  laboratory. 
The  animals  appeared  not  to  be  disturbed  by  the  injections  in 
so  far  as  weight,  temperature,  and  demeanor  indicated,  and  nothing 
of  a  positive  nature  was  learned  from  the  experiments. 

On  May  6,  5  c.c.  of  defibrinated  blood,  22  hours  old,  from  the 
patient,  Cortsen,  were  injected  intravenously  into  a  rabbit  weigh- 
ing about  two  kilos.  No  disturbance  was  noted  for  four  or  five 
days,  when  the  temperature  rose  to  104.8°,  but  .remained  above 
normal  for  no  more  than  two  or  three  days.  The  experiment  was 
not  considered  satisfactory. 

experiments  with  guinea-pigs. 

Turning  to  the  guinea-pig,  a  supply  of  which  had  been  received 
in  time  for  inoculations  with  the  Cortsen  blood,  astonishing  and 


28o  Contributions  to  Medical  Science 

strikingly  positive  results  were  obtained.  On  May  5,  when  the 
Cortsen  blood  was  six  and  one-half  hours  old,  one  guinea-pig  was 
given  3  c.c.  and  another  5  c.c.  intraperitoneally.  One  of  the 
animals  died  in  7  days  and  the  other  in  1 1  days.  It  was  necessary 
for  me  to  be  absent  from  Missoula  for  two  or  three  days  following 
the  inoculations,  but  on  returning  I  found  in  both  a  temperature 
which  ranged  between  104°  and  106°  F.  After  the  fifth  day  both 
animals  became  emaciated  rapidly,  and  for  from  36  to  48  hours 
before  death  the  temperature  was  subnormal.  In  guinea-pig  i, 
the  scrotum  and  testicles  became  enormously  swollen,  and  on  the 
sixth  day  the  skin  of  the  scrotum  was  occupied  by  large  dark- 
colored  hemorrhages  of  irregular  outline.  Subsequent  experience 
showed  that  this  sweUing  of  the  testicles  and  scrotum  and  the 
hemorrhage  into  the  skin  of  the  scrotum  is  the  most  characteristic 
sign  of  the  disease  as  produced  in  the  male  guinea-pig.  Cultures 
of  the  Cortsen  blood,  and  also  of  the  heart's  blood  of  the  guinea- 
pigs  after  their  death,  remained  free  from  discoverable  growth. 

A  second  generation  of  three  guinea-pigs  was  inoculated  with 
the  heart  blood  and  splenic  emulsion  from  the  two  animals  which 
had  died.  Of  the  second  generation  one  died,  having  the  character- 
istic scrotal  hemorrhage  and  swelling,  and  the  other  two  recovered 
after  a  course  of  high  fever  and  emaciation.  From  the  fatal  case 
inoculations  were  made  into  a  third  generation,  the  members  of 
which  did  not  become  noticeably  sick.  In  all  fatal  cases  cultures 
of  the  blood  on  plates  gave  no  visible  growth. 

Although  these  experiments  can  leave  no  reasonable  doubt 
as  to  the  actual  transference  of  the  infection,  further  work  seemed 
to  be  threatened  by  the  inability  to  keep  the  infection  going 
indefinitely  in  the  guinea-pig. 

On  May  20,  blood  from  the  patient,  Porter,  was  inoculated 
into  two  male  guinea-pigs,  one  of  which  died  in  seven  and  the  other 
in  eight  days,  both  cases  running  t}^ical  febrile  courses,  and 
showing  the  changes  in  the  scrotum  already  mentioned.  Cultures 
of  the  Porter  blood  and  of  the  heart's  blood  of  the  fatal  inoculations 
remained  sterile. 

A  second  generation  of  three  male  guinea-pigs  was  injected  from 
each  of  the  fatal  inoculations  of  the  first  generation.     The  two 


The  Study  of  "Rocky  Mountain  Spotted  Fever"    281 

members  of  the  second  generation  became  very  sick,  running 
typical  courses  of  fever  which  reached  105.7°  and  105. 8°,  but  re- 
covery appeared  to  be  complete  on  the  tenth  day  in  each  case,  at 
which  time  the  temperature  had  returned  to  the  normal.  Here,  too, 
attempts  to  perpetuate  the  infection  by  direct  transference  failed. 
On  June  11  blood  from  the  patient,  Bradley,  whom  I  discovered 
accidently  near  Stevensville,  was  utilized  partly  in  repetition 
of  previous  experiments,  but  chiefly  to  determine  the  filterability 
of  the  virus  and  its  distribution  among  the  constituents  of  the 
blood.  For  this  purpose  a  sufiicient  quantity  of  the  blood  was 
centrifugated  for  45  minutes  at  an  estimated  speed  of  1,500 
revolutions.  The  serum  was  drawn  off  and  6  c.c.  were  injected 
intraperitoneally  into  a  guinea-pig.  An  additional  amount  of 
6  c.c.  was  passed  through  a  small  Berkefeld  filter  under  low  pres- 
sure, the  total  amount  after  the  filter  was  washed  out  with  salt 
solution  being  between  10  and  12  c.c.  This  amount  was  con- 
sidered too  large  for  a  single  injection,  hence  it  was  given  to  an- 
other guinea-pig  in  two  injections  separated  by  an  interval  of  two 
hours.  The  erythrocytes,  and,  of  course,  leukocytes,  which 
remained  after  centrifugation,  were  now  washed  three  times  with 
sterile  salt  solution,  raised  to  the  original  volume  by  means  of  the 
same  solution,  and  6  or  7  c.c.  injected  intraperitoneally  into  another 
guinea-pig.  To  complete  this  experiment,  the  unaltered  defi- 
brinated  blood  was  injected  intraperitoneally  into  another  guinea- 
pig.  Without  giving  details  as  to  the  course  of  the  disease  in  these 
animals,  the  results  were,  briefly,  as  follows:  Fresh  defibrinated 
blood,  washed  corpuscles,  and  unfiltered  serum  produced  typical 
and  fatal  infections  in  the  guinea-pigs  inoculated  with  them, 
whereas  the  filtered  serum  had  no  discoverable  effect,  the  animal 
at  no  time  showing  a  temperature,  even  up  to  the  present  time. 

RESULTS   IN  EXPERIMENTS. 

The  results  of  these  experiments  were  somewhat  surprising, 
since  the  failure  to  recognize  the  organism  microscopically  had 
suggested  the  probability  that  it  was  of  exceedingly  minute  size 
and  might  be  readily  filterable.  I  would  not  assume  that  filtration 
is  impossible,  but  simply  state  that  it  did  not  pass  through  the 


282  Contributions  to  Medical  Science 

filter  under  the  condition  of  experiment.  These  experiments 
prove,  then,  that  the  condition  produced  in  the  guinea-pig  is  an 
infection  and  not  an  intoxication,  for  otherwise  the  filtered  serum 
undoubtedly  would  be  as  toxic  as  the  unfiltered.  In  view  of  the 
infectiousness  of  the  serum  they  also  prove  that  the  infecting 
agent  is  far  from  being  an  exclusive  corpuscular  parasite;  and 
they  suggest  very  strongly  that  the  organism  is  of  such  size 
that  its  ultimate  and  positive  recognition  with  the  microscope 
may  be  hoped  for  reasonably.  It  is  recognized  that  these  results 
do  not  agree  positively  or  decisively  against  the  piroplasma  theory, 
since  piroplasmata  may  well  be  in  the  serum  as  in  the  erythrocytes. 
But  they  do  seem  to  throw  some  doubt  on  this  theory,  since  the 
serum  was  almost  as  virulent  as  the  erythrocytes.  On  the  other 
hand,  it  cannot  be  argued  that  because  the  washed  corpuscles 
were  highly  infectious  that  the  erythrocytes  actually  are  infected. 
One  could  as  readily  assume  that  the  organisms  were  in  the  leuko- 
cytes, or  that  they  were  entirely  extracellular,  a  certain  proportion 
of  them  having  been  thrown  down  in  the  centrifuge  together 
with  the  corpuscles. 

Another  important  point  brought  out  by  these  inoculations 
with  the  Bradley  blood  is  the  fact  that  the  guinea-pig,  at  least 
in  some  instances,  suffers  from  a  more  or  less  extensive  macular 
and  confluent  eruption  which  is  in  addition  to  the  scrotal  hemor- 
rhage. This  had  been  missed  in  the  earlier  experiments,  partly 
because  of  its  unsuspected  distribution.  After  observing  that 
such  an  eruption  occurred  chiefly  on  the  dorsal  aspect  of  a  monkey 
inoculated  by  Dr.  King,  being  entirely  absent  from  the  abdomen 
and  chest,  I  shaved  the  entire  skin  of  a  guinea-pig  which  had  died 
of  the  disease,  and  found  the  dorsal  and  lateral  aspects  of  the 
body,  face,  and  extremities  marked  by  many  reddish  macules, 
whereas  over  the  face  and  buttocks  there  were  confluent  hemor- 
rhages. Subsequent  observation  on  other  guinea-pigs  shows 
that  this  eruption  is  of  frequent,  though  perhaps  not  constant, 
occurrence. 

experiments  on  monkeys. 

I  ordered  monkeys  as  soon  as  I  had  decided  to  begin  animal 
inoculations.    The   season   of   the   disease  being  short   and   the 


The  Study  of  "Rocky  Mountain  Spotted  Fever"    283 

situation  more  or  less  critical,  it  was  thought  best  not  to  jeopardize 
progress  by  neglecting  the  use  of  an  animal  so  closely  related  to 
man.  After  unfortunate  delays  and  misunderstandings  these 
animals  reached  me  in  time  for  inoculation  with  the  blood  of  the 
patient,  Porter. 

On  May  20  a  healthy  and  fairly  large  Rhesus,  weighing  about 
three  kilos,  received  intravenously  10  c.c.  of  defibrinated  blood 
from  the  patient,  Porter.  The  blood  at  this  time  was  about  28 
hours  old,  this  period  representing  the  time  required  to  transport 
it  from  Hamilton,  50  miles  away.  During  this  time  the  blood 
had  been  kept  at  outdoor  temperature,  about  15°  C.  On  the 
evening  of  the  second  day  the  temperature  of  the  animal  rose  to 
104.6°  by  rectum,  and  from  this  time  until  the  tenth  day  it  ranged 
from  103.1°  to  105.3°.  Ori  the  tenth  day  it  fell  rather  suddenly 
to  loi .  1°,  and  the  animal  gradually  regained  his  former  healthy 
condition.  From  the  fourth  to  the  eighth  day  he  appeared  very 
sick,  was  extremely  weak,  ate  very  little,  the  face  was  cyanotic 
and  the  conjunctivae  were  infected.  The  cyanosis  and  injected 
conjunctivae  are  recognized  as  characteristic  symptoms  of  spotted 
fever  as  the  disease  occurs  in  man.  The  duration  of  the  sickness 
also  approximated  that  seen  in  man.  The  incubation  period 
was  shorter,  however,  as  in  the  guinea-pig. 

When  the  disease  is  fatal  in  man,  death  usually  occurs  on  from 
the  seventh  to  the  ninth  day;  and,  although  patients  occasionally 
die  after  having  weathered  14  or  15  days,  one  who  survives  the 
tenth  is  given  a  guarded  favorable  prognosis.  The  most  character- 
istic sign  of  the  disease  as  it  is  seen  in  man,  i.e.,  the  generalized 
reddish  macular  eruption,  was  not  observed  in  this  animal.  Dur- 
ing his  sickness  the  eruption  was  sought  for  principally  on  the  chest, 
abdomen,  arms,  and  legs,  where  there  was  the  least  hair,  and  the 
skin  in  some  places  was  of  a  transparent  pink  color.  The  absence 
of  the  eruption  does  not,  under  the  circumstances,  render  the 
diagnosis  of  spotted  fever  untenable.  Those  who  have  had  the 
greatest  experience  with  the  disease  in  man  recognize  a  mild  type 
in  which  the  characteristic  eruption  is  absent.  In  such  cases,  the 
diagnosis  rests  chiefly  on  the  sluggish  circulation,  general  cyanosis, 
reddened  conjunctivae,  drowsiness,  and  lack  of  complaint  on  the 


284  Contributions  to  Medical  Science 

part  of  the  patient  in  spite  of  retained  intelligence,  the  course  and 
duration  of  the  fever,  slow  convalescence,  and  the  season  of  the 
year  in  which  the  disease  occurs.  Furthermore,  those  who  are 
familiar  with  experimental  inoculations  in  animals  have  long 
since  recognized  the  fact  that  in  many  diseases,  desirable  as  the 
result  is,  one  may  not  be  able  to  obtain  in  an  animal  a  complete 
duplication  of  all  the  symptoms  seen  in  man,  in  spite  of  exquisite 
susceptibility  on  the  part  of  the  animal.  The  receptor  theory  of 
Ehrlich  has  rendered  such  variations  altogether  intelligible. 

My  second  inoculation  of  the  monkey  was  made  on  June  11 
with  the  defibrinated  blood  of  the  patient,  Bradley.  This  patient 
was  discovered  by  the  merest  accident,  on  June  9,  near  Stevensville, 
where  I  had  gone  for  the  purpose  of  exploring  an  infected  district 
with  Dr.  Fessler.  On  the  following  day  from  60  to  70  c.c.  of 
blood  were  drawn  from  a  vein  of  the  arm  by  means  of  sterile  appara- 
tus kindly  loaned  by  Dr.  Brice,  the  attending  physician.  The 
patient  was  in  the  tenth  day  of  the  disease.  After  defibrination 
the  blood  was  placed  on  ice  until  the  following  day,  when  it  was 
possible  to  return  to  Missoula. 

The  animal  inoculated  was  a  small  female  Rhesus,  weighing 
less  than  two  kilos  and  apparently  perfectly  healthy.  Eight 
c.c.  of  the  blood  were  injected  intraperitoneally.  The  animal 
ran  a  course  of  fever  similar  to  that  of  the  first  monkey,  but  died 
on  the  ninth  day,  after  a  short  period  of  subnormal  temperature. 
In  addition  to  the  cyanosis  of  the  face  and  ears,  noted  in  the  first 
monkey,  the  hairless  skin  of  the  perineum  became  brilliantly  red, 
although  at  no  time  were  hemorrhages  detectable.  The  skin  of 
the  abdomen,  chest,  arms,  and  legs  remained  free  from  spots  or 
hemorrhages.  No  cause  of  death,  other  than  spotted  fever,  was 
found  at  the  autopsy,  no  peritonitis  had  followed  the  injection,  and 
a  plate  culture  of  the  heart's  blood  showed  no  visible  growth 
after  five  days  of  incubation. 

A  second  monkey  and  three  guinea-pigs  were  inoculated  with 
the  spleen  and  blood  of  the  dead  animal  as  soon  as  possible  after 
death.  One  guinea-pig  died  of  peritonitis  following  perforation 
of  the  bowel  with  the  needle.  The  other  two  are  at  present  running 
temperatures  which  correspond  to  that  seen  in  previous  inocula- 


The  Study  of  "Rocky  Mountain  Spotted  Fever"    285 

tions  of  the  guinea-pig,  and  in  one  the  scrotum  shows  the  character- 
istic enlargement.  The  monkey  of  the  second  generation  has  a 
temperature  which  has  gone  as  high  as  106 . 7°,  the  perineum  shows 
the  redness  seen  in  the  monkey  of  the  first  generation,  and  the 
hemorrhagic  eruption  has  appeared  on  the  scrotum,  buttocks, 
legs,  back,  etc.  The  appearance  of  these  phenomena  in  the 
second  generation  of  animals  leaves  no  doubt  as  to  the  success  of 
the  inoculation  with  the  blood  of  the  patient,  Bradley. 

I  may  refer  here  to  a  most  gratifying  result  obtained  by  Passed 
Assistant-Surgeon  W.  W.  King,  of  the  Public  Health  and  Marine 
Hospital  Service.  Up  to  the  time  of  the  inoculations  with  the 
Bradley  blood.  Dr.  King  had  confined  himself  to  the  microscopic 
study  of  the  blood.  He  had  been  no  more  successful,  however,  in 
this  search  than  I,  and,  having  obtained  a  monkey  and  some 
guinea-pigs,  I  readily  assented  to  his  proposal  that  he  repeat  some 
of  my  experiments  with  the  hope  of  confirming  them.  For  this 
purpose  I  shared  with  him  the  blood  of  the  patient,  Bradley.  We 
drew  lots  for  the  route  of  inoculation,  the  subcutaneous  falling 
to  Dr.  King  and  the  intraperitoneal  to  me,  as  stated  above.  The 
monkey  inoculated  by  Dr.  King  died  two  days  later  than  mine 
and  showed  a  marked  hemorrhagic  condition  of  the  skin  of  the 
scrotum  and  penis  and  the  flaming  erythema  of  the  perineum 
seen  in  my  monkeys,  together  with  a  mixed  macular  and  confluent 
eruption,  hemorrhagic  in  character,  which  was  distributed  chiefly 
over  the  external  aspects  of  the  arms,  legs,  buttocks,  and  back. 
The  face  and  ears  were  extremely  cyanotic.  No  exanthem  could 
be  discovered  on  the  chest  and  abdomen.  A  guinea-pig  also 
inoculated  by  Dr.  King  with  the  Bradley  blood  ran  a  typical 
course,  presenting  the  enlarged  and  hemorrhagic  scrotum,  the 
exanthem  already  observed  by  me,  and  in  other  respects  confirm- 
ing the  results  of  my  inoculations. 

White  rats  and  mice  arrived  so  late  that  they  could  be  used  in 
but  one  case;   the  disease  was  not  reproduced  in  them. 

SUMMARY. 

The  essential  anatomic  findings  in  the  guinea-pig  at  autopsy 
consist  of  the  cutaneous  phenomena  described,  the  enlarged  and 


286  Contributions  to  Medical  Science 

hemorrhagic  scrotum  in  the  males  and  some  swelling  of  the  testicles 
with  pronounced  congestion  of  the  epididymis,  retention  of  urine, 
distension  of  the  seminal  vesicles,  congestion  of  the  kidneys  and 
suprarenals,  swelling  and  congestion  of  the  spleen  and  liver,  and  a 
right  heart  and  venous  system  which  are  enormously  engorged 
with  blood.  No  meningitis  nor  localized  inflammations  have  been 
observed,  except  in  two  instances  in  which  the  skin  of  the  swollen 
scrotum  had  become  gangrenous,  with  a  consequent  staphylococcus 
infection  of  the  underlying  cellular  tissue. 

In  the  one  monkey  which  has  gone  to  autopsy  so  far,  nothing 
was  found  macroscopically,  in  addition  to  the  cutaneous  phenomena, 
except  a  congestion  of  the  parenchymatous  organs. 

That  spotted  fever  has  been  transmitted  to  the  guinea-pig  in 
my  experiments  is  shown  by  the  following  data:  The  fever,  dura- 
tion, and  cutaneous  phenomena  resemble  very  closely  these  condi- 
tions as  seen  in  man.  It  can  be  transmitted  into  the  third  genera- 
tion by  direct  inoculation  from  one  animal  to  another,  but  has  not 
been  carried  beyond  this  point  up  to  the  present.  The  condition 
produced  has  been  an  actual  infection  rather  than  a  transferred 
intoxication,  because  it  can  be  passed  through  the  second  genera- 
tion of  animals,  and  because  the  filtered  serum  which  would  cer- 
tainly contain  any  soluble  toxins  which  might  be  present  causes  no 
disturbance.  In  further  confirmation  of  the  statements  just 
made  are  the  facts  that  no  other  cause  of  death  has  been  found, 
and  that  it  has  not  been  possible  to  cultivate  any  micro-organism 
from  the  heart's  blood  in  fatal  cases. 

For  similar  reasons  it  is  believed  that  the  transmission  to  the 
monkey  cannot  be  disputed. 

I  realize  that  the  material  on  which  these  conclusions  are  based 
is  rather  scant,  but  the  experiments  have  been  rigorously  con- 
trolled, and  I  feel  that  they  are  safe. 

The  hope  is  still  entertained  that  it  may  be  possible  to  keep 
the  infection  alive  in  experiment  animals  by  suitable  manipula- 
tions in  order  that  many  other  problems  bearing  on  the  disease 
may  be  taken  up  properly.  I  am  at  present  trying  to  do  this  by 
alternating  the  infection  between  the  monkey  and  the  guinea-pig, 
with  the  hope  that  the  character  of  the  \-irus  may  be  so  altered 


The  Study  of  "Rocky  Mountain  Spotted  Fever"    287 

that  the  infection  may  be  passed  directly  from  one  guinea-pig  to 
the  other  indefinitely.  With  this  result  in  hand  attempts  toward 
the  production  of  a  vaccine  may  reasonably  be  made. 

The  discussion  of  other  features  of  the  disease  will  be  reserved 
for  the  present.  I  shall  only  refer  to  the  theory  of  transmission 
by  means  of  the  wood-tick,  by  expressing  my  disagreement  with 
the  conclusion  of  Stiles  that  the  tick  theory  falls  with  the  piro- 
plasma  theory.  The  tropical  "tick  fever"  goes  to  show  that 
ticks  may  harbor  and  transmit  pathogenic  parasites  other  than 
piroplasmata. 

For  the  privileges  of  the  laboratory  of  the  Northern  Pacific 
Hospital  and  of  the  hospital  itself,  I  am  under  the  greatest  obliga- 
tion to  Dr.  W.  E.  Spottswood,  surgeon-in-chief,  without  whose 
active  interest  much  of  the  work  of  this  season  could  not  have  been 
undertaken.  I  also  express  my  thanks  to  Dr.  Chowning,  since 
I  profited  greatly  by  his  previous  experience  in  the  field,  and  to 
Dr.  King,  who,  after  the  departure  of  Dr.  Chowning,  gave  me 
the  most  generous  assistance  in  the  inoculation  of  the  animals. 

The  contributions  of  the  Montana  State  Board  of  Health  and 
of  Missoula  and  Ravalli  counties,  through  their  commissioners, 
are  gratefully  acknowledged. 


THE  TRANSMISSION  OF  ROCKY  MOUNTAIN  SPOTTED 
FEVER    BY    THE    BITE    OF    THE   WOOD-TICK 
(DERMACENTOR  OCCIDENTALIS).^ 

H.     T.     RiCKETTS. 

In  The  Journal,  July  7  (see  p.  278  of  this  volume),  I  reported 
briefly  my  success  in  transmitting  Rocky  Mountain  spotted  fever 
to  the  guinea-pig  and  monkey.  With  these  results  in  hand,  it 
at  once  became  apparent  that  a  means  had  been  provided  by 
which  to  test  the  theory  of  transmission  by  the  wood-tick,  a 
theory  which  was  primarily  advanced  by  Wilson  and  Chowning. 
It  was  my  first  aim  to  establish,  beyond  the  opportunity  of 
doubt,  the  susceptibility  of  the  animals  mentioned,  and  it  was 
only  after  this  susceptibiUty  had  been  determined  by  inocula- 
tion from  two  different  patients  that  the  study  of  the  tick  as 
an  intermediate  host  for  the  parasite  was  taken  up. 

On  June  19  a  small  female  tick  was  placed  at  the  base  of  the 
ear  of  "Bradley"  guinea-pig  i.  This  guinea-pig  had  been  inocu- 
lated intraperitoneally  on  June  11  with  three  cubic  cm.  of  defi- 
brinated  blood  from  the  patient,  Bradley,  and  died  with  character- 
istic symptoms  on  June  23.  After  having  fed  on  the  infected 
guinea-pig  for  two  days,  the  tick  was  removed,  placed  in  a  venti- 
lated pill-box  for  two  days  more,  and  on  June  23  was  attached  to 
the  base  of  the  ear  of  a  female  guinea-pig  weighing  300  gms.  After 
an  incubation  period  of  three  and  one-half  days,  the  temperature 
of  the  animal  rose  to  104.2,  gradually  ascended  to  106.4,  near 
which  point  it  remained  for  seven  days,  when  it  gradually  returned 
to  normal.  The  bite  of  the  tick  in  this  instance  seemed  to  be 
very  virulent,  the  whole  ear  becoming  very  much  swollen,  indurated 
and  cyanotic,  and  a  slough  almost  one-half  inch  in  diameter, 
which  developed  in  the  vicinity  of  the  bite,  eventually  separated. 

'  This  work  has  been  done  in  part  under  a  grant  made  by  the  American  Medical  Association  through 
the  Committee  on  Scientific  Research.     From  Jour.  Am.  Med.  Assn.,  1906,  47,  p.  358. 

288 


July   I,  A.M. 

104.6. 

July   2,   A.M. 

105.2. 

July  3,  A.M., 

104.7; 

P.M., 

lOS- 

July  4,  A.M., 

104.7; 

P.M., 

103.1 

July  5,  A.M., 

103.8; 

P.M., 

103.9 

July  6,  A.M., 

103  4; 

P.M., 

103  S 

July  7,  A.M., 

102.5. 

Recovery. 

Transmission  of  Spotted  Fever  by  Wood-Tick       289 

The  animal  exhibited  the  following  course  of  fever: 

June  23,  A.M.,  102.3;  P-^-,   102.2. 

June  24,  A.M.,  loi  .Q. 

June  25,  A.M.,  102.8;  P.M.,  103.9 

June  26,  A.M.,  102.4;  P.M.,  103.6 

June  27,  A.M.,  102.5;  P-^-,   104.2 

June  28,  A.M.,  106.4;  P-M.,  105.2 

June  29,  A.M.,  105.6;  P.M.,  105.8 

June  30,  A.M.,  104.8;  P.M.,  106. 1 

Seven  days  after  inoculation,  the  external  genitalia  became 
swollen  and  congested,  and  this  condition  became  more  marked 
until  the  twelfth  day,  when  it  began  to  subside.  At  no  time  were 
distinct  hemorrhages  in  the  genitalia  or  other  parts  of  the  skin 
to  be  seen.  The  course  of  the  disease  was  exactly  similar  to  that 
seen  in  a  number  of  other  guinea-pigs  which  ran  a  severe  course, 
with  eventual  recovery.  The  absence  of  a  hemorrhagic  condition 
in  the  skin,  or  of  a  discoverable  roseolar  eruption,  throws  no  doubt 
on  the  success  of  the  transmission,  since,  as  shown  by  other  experi- 
ments, a  perceptible  eruption  does  not  appear  invariably  in  spotted 
fever  as  it  is  produced  in  the  guinea-pig  experimentally. 

It  was  not  possible  to  attempt  the  transmission  of  the  disease 
from  this  animal  to  others,  because  of  the  lack  of  guinea-pigs  at 
the  time. 

As  controls  I  have  two  experiments  in  which  uninfected  ticks 
fed  on  normal  guinea-pigs  without  causing  a  rise  in  temperature; 
furthermore,  two  guinea-pigs  which  lived  for  two  weeks  in  the 
box  occupied  by  the  infected  animal  showed  no  abnormal  tem- 
perature, a  fact  which  argues  against  the  transmission  of  the  infec- 
tion by  mere  association  with  excretions  of  infected  animals. 

The  result  of  this  experiment  brought  very  forcibly  to  my  mind 
the  probable  part  which  the  tick  plays  in  the  infection  of  man 
and  shows  the  necessity  of  repetition  of  the  work  with  a  more 
abundant  material.  In  view  of  the  result  which  I  had  obtained 
I  was  not  surprised  to  note  the  recent  report  of  Dr.  King,  of  the 
U.S.  Public  Health  and  Marine  Hospital  Service,  who,  starting 
with  material  which  I  had  given  him,  accomplished  transmission 
in  the  same  manner. 

Hasty  conclusions  as  to  the  question  of  tick  transmission  in 


290  Contributions  to  Medical  Science 

relation  to  the  infection  of  man  are,  by  all  means,  to  be  avoided 
until  such  a  time  as  the  experiments  can  be  repeated  and  the  life- 
history  of  the  infection  worked  out  more  thoroughly.  This  phase 
of  the  subject,  in  common  with  others,  is  being  studied  by  me,  the 
infection  still  being  maintained  in  animals  for  these  purposes. 

It  is  hoped  that  some  questions  may  be  settled  satisfactorily 
before  the  advent  of  the  disease  next  year.  At  all  events  the 
knowledge  so  far  gained  may  well  be  taken  into  account  in  institut- 
ing prophylactic  measures  against  the  disease. 


FURTHER   OBSERVATIONS   ON   ROCKY   MOUNTAIN 

SPOTTED   FEVER  AND   DERMACENTOR 

OCCIDENTALIS.^ 

H.     T.     RlCKETTS. 

(From  the  Pathological  Laboratory  oj  the  University  of  Chicago.) 

In  view  of  the  brief  season  during  which  Rocky  Mountain 
spotted  fever  prevails,  uninterrupted  study  of  the  disease  is  con- 
ditioned on  the  maintenance  of  the  infection  in  experiment  animals. 

In  a  previous  communication^  (p.  278)  my  inability  to  preserve 
the  disease  by  inoculation  from  guinea-pig  to  guinea-pig  was  referred 
to,  and  the  hope  was  expressed  that  alternation  of  the  infection 
between  the  monkey  and  the  guinea-pig  might  result  in  the  preser- 
vation of  the  virus. 

This  experiment  has  been  entirely  successful  and  Table  i  illus- 
trates the  life  history  of  the  virus  since  it  was  obtained  from  the 
patient,  Bradley. 

TABLE  I. 


Number  of  Animal 

Date 

Source,  and  Material  Inoculated 

Monkey  I 

June  11 

Patient,  Bradley;  defibrinated  blood 

Monkey  II 

June  20 

Monkey  I;  defibrinated  blood 

Guinea-pig  12 

June  27 

Monkey  II;  fresh  heart's  blood 

Monkey  IV 

July    7 

Guinea-pig  16;  blood  and  organs 

Guinea-pig  16 

July  14 

Monkey  IV;  defibrinated  blood 

Monkey  V 

July  22 

Guinea-pig  16;  blood  and  organs 

Guinea-pig  17 

July  28 

Monkey  V;  defibrinated  blood 

Monkey  VI 

August    3 

Guinea-pig  17;  blood  and  spleen 

Guinea-pig  2  s 

August  16 

Monkey  VI;  blood  and  serum 

Monkey  VIII 

August  23 

Guinea-pig  25;  blood  and  organs 

Guinea-pig  36 

August  30 

Monkey  VIII;  spleen 

Monkey  IX 

September  6 

Guinea-pig  36;  blood  and  organs 

All  infected  animals  have  run  identical  courses  of  fever,  which 
are  typical  in  this  respect,  that  an  incubation  period  of  from  two 
to  four  days  intervenes  between  the  time  of  inoculation  and  the 
onset  of  the  regular  febrile  course.     Commonly  a  moderate  rise 

■  This  work  has  been  done  under  a  grant  made  by  the  American  Medical  Association  through  the 
Committee  on  Scientific  Research,  and  with  the  aic  of  Missoula  and  Ravalli  counties,  Montana,  and  of 
the  Montana  State  Board  of  Health.     From  Jour.  Am.  Med.  Assn.,  IQ06,  47,  p.  1067. 

'  "The  Study  of  Rocky  Mountain  Spotted  Fever  (Tick  Fever?)  by  Means  of  Animal  Inoculations," 
ibid.,  1906,  47.  P-  a- 

291 


292  Contributions  to  Medical  Science 

in  temperature  follows  immediately  on  the  inoculation  and  persists 
for  one  or  two  days,  then  falls  near  to  normal  for  one  or  two  days 
more,  when  the  customary  course  sets  in.  This  initial  or  primary 
fever  probably  is  caused  by  intoxication  from  the  large  amount 
of  material  which  is  frequently  injected.  When  a  smaller  amount 
of  blood  or  emulsion  of  tissue  is  used  this  initial  fever  has  not 
appeared. 

Several  facts  are  relied  on  in  concluding  that  the  disease  trans- 
ferred by  these  inoculations  is  spotted  fever  rather  than  some  other 
infection  which  may  have  crept  in  during  repeated  passage.  In 
harmony  with  the  experience  which  was  met  in  making  cultures 
from  man,  no  growth  is  obtained  from  the  blood  of  infected  ani- 
mals, provided  the  cultures  are  made  at  a  time  when  agonal  or 
postmortem  invasion  by  common  bacteria  has  not  taken  place. 
If  plates  are  inoculated  with  the  heart's  blood  at  the  instant  of 
death,  it  is  customary  to  obtain  no  visible  growth  on  ordinary 
media,  yet  this  same  blood  proves  infectious  for  other  animals. 
Again,  the  rather  frequent  development  of  a  hemorrhagic  eruption 
in  the  monkey  or  guinea-pig,  a  manifestation  which  is  identical 
in  appearance,  if  not  in  distribution,  with  that  seen  in  man,  speaks 
for  spotted  fever  rather  than  some  other  infection.  This  eruption 
is  much  more  constant  in  the  monkey  than  in  the  guinea-pig.  In 
both  animals  it  begins  on  the  scrotum  and  foreskin.  In  the  guinea- 
pig  it  may  be  found  on  no  other  part  of  the  body,  although  it  can 
be  seen  as  a  more  or  less  generalized  eruption  occasionally.  The 
external  genitals  of  the  female  guinea-pig  commonly  become 
swollen  and  congested;  recently,  however,  extensive  hemorrhages 
have  occurred  in  the  skin  of  the  labia  in  a  number  of  females. 
But  one  female  monkey  has  been  used,  and  in  her  no  cutaneous 
hemorrhages  were  discovered.  It  will  readily  be  understood  that 
the  detection  of  a  pink  roseolar  eruption  in  either  the  monkey  or 
the  guinea-pig  is  difficult.  In  the  guinea-pig  it  can  be  recognized 
with  certainty  only  in  white-skinned  animals.  In  both  animals 
the  hemorrhagic  eruption,  when  it  appears,  is  a  late  manifestation 
and  it  would  seem  to  be  related  in  some  way  to  the  virulence  and 
duration  of  the  infection.  The  disease  may  be  so  virulent  that  the 
animal  dies  before  the  eruption  would  appear,  or  so  mild  that  it 


Spotted  Fever  and  Dermacentor  Occidentalis       293 

does  not  occur.  The  appearance  of  the  hemorrhages  does  not 
necessarily  presage  a  fatal  issue ;  two  monkeys  and  several  guinea- 
pigs  which  had  extensive  hemorrhages  into  the  skin  have  recovered. 

A  third  fact  which  stamps  this  experimental  disease  as  spotted 
fever  consists  of  the  identity  of  the  course  of  fever  with  that  pro- 
duced in  the  animals  by  direct  inoculation  with  human  blood. 

The  essential  anatomic  changes — enlarged  spleen  and  lymph 
glands,  congested  kidneys,  etc. — are  identical  with  those  seen  in  the 
animals  first  inoculated,  and  correspond  with  the  changes  found  in 
man. 

There  seems  to  be  no  particular  difficulty  involved  in  perpetu- 
ating the  infection,  except  in  the  matter  of  expense.  Duplicate 
inoculations  should  be  made  and  the  blood  or  organs  used  for 
inoculation  may  be  taken  at  any  time  after  the  fever  has  reached 
its  high  point.  To  await  the  death  of  the  animal  before  making 
the  transfer  is  to  risk  the  loss  of  the  culture ;  the  animal  may  recover. 

THE   DISTRIBUTION   OF   THE   VIRUS   IN   THE   BODY   OF   THE   INFECTED 

ANIMAL. 

In  my  first  experiments  the  infectiousness  of  the  blood,  in 
spotted  fever,  was  demonstrated.  It  would  naturally  be  inferred 
that  the  various  organs  are  infectious  at  least  to  the  degree  in 
which  they  contain  blood.  On  this  account  one  would  reasonably 
expect  the  spleen  to  be  infectious  and  this  has  proven  true  in  a 
number  of  inoculations.  The  testicles  have  been  used  for  inocu- 
lation a  number  of  times.  It  was  thought  that  the  testicles  might 
be  a  point  of  predilection  for  the  virus,  in  view  of  the  severe  hemor- 
rhages which  often  occur  in  close  proximity  to  them,  i.e.,  in  the 
scrotum.  They  were  shown  to  be  infectious  in  several  instances, 
yet  their  virulence  was  not  so  great  as  to  indicate  any  special  con- 
centration of  the  virus  at  this  point.  The  scrotal  hemorrhages 
may  have  no  relation  to  the  amount  of  infection  of  the  testicles. 

The  distribution  of  the  virus  among  the  organs  of  an  infected 
monkey  (VIII)  was  recently  determined.  The  animal  was  exsan- 
guinated from  the  carotid  to  deprive  the  organs  of  as  much  blood 
as  possible.  Each  organ  was  then  ground  with  sterile  sand  and 
suspended  in  physiologic  salt  solution.     Identical  weights  of  the 


294 


Contributions  to  Medical  Science 


different  organs  were  not  used,  but  approximately  the  same  mass 
was  employed  in  each  case.  A  further  attempt  at  uniformity  was 
made  by  rendering  the  different  emulsions  of  approximately  the 
same  density.  Five  cubic  centimeters  of  each  emulsion  were 
injected  intraperitoneally  into  guinea-pigs,  which  varied  in  weight 
from  350  to  550  grams.     Table  2  shows  the  results. 


TABLE  2. 
The  Infectiousness  of  Different  Organs. 


Number 

Matter  Inoculated 

Result 

35 

36 

37 

38 

39 

40 

41 

42 

Mesenteric,  inguinal  and  axillary  lymph 
glands 

Spleen 

Bone-marrow 

Testicles 

Liver 

Kidney 

Medulla  oblongata 

Cerebral  cortex  and  adjacent  white  mat- 
ter 

Death  in  i8  days 

Death  in  7  days 
Death  in  6  days 
Death  in  15  days 
Death  in  8  days 
Death  in  7J  days 
Recovered 
Death  in  i2i  days 

In  the  fatal  cases  cultures  of  the  heart's  blood  were  sterile 
except  in  the  case  of  Guinea-pig  37,  in  which  a  few  colonies  of  a 
staphylococcus  developed.  The  technical  difficulties  in  preparing 
the  bone-marrow  increased  the  opportunities  for  infection.  It  is 
thought  very  probable,  however,  that  the  animal  died  of  spotted 
fever  because  of  the  duration  of  the  course. 

Those  organs  seemed  to  be  the  most  infectious  which  contained, 
naturally,  the  most  blood,  i.e.,  the  liver,  spleen,  and  bone-mar- 
row (?).  In  these  organs  exsanguination  still  leaves  rather  large 
quantities  of  blood.  The  kidney,  however,  was  fully  as  infectious 
as  the  organs  which  were  much  richer  in  blood.  It  is  possible  that 
the  kidney  becomes  the  repository  of  a  larger  mass  of  micro-organ- 
isms, by  virtue  of  its  excretory  function.  It  is  realized  that  this 
experiment  does  not  give  an  exact  measurement  of  the  degree  of 
infection  of  the  different  organs,  and  that  the  results,  at  the  best, 
can  be  considered  no  more  than  approximate. 

In  another  experiment  an  attempt  was  made  to  determine  the 
minimum  fatal  dose  of  the  blood  of  the  infected  monkey,  the  blood 
being  taken  six  days  after  inoculation.  The  injections  were  intra- 
peritoneal (Table  3). 


Spotted  Fever  and  Dermacentor  Occidentalis       295 

Plate  cultures  of  the  heart's  blood  were  sterile  in  all  cases.  In 
this  experiment  the  minimum  fatal  dose  of  the  blood  of  the  infected 
monkey  on  the  sixth  day  of  the  disease  was  in  the  vicinity  of  o .  5 
c.c.     Guinea-pig  30,  which  received  this  dose,  died  on  the  four- 

TABLE  3. 
Virulence  of  the  Blood  of  the  Infected  MoNKj;y. 

Result 

Death  in  14  days 
Death  in  7i  days 
Death  in  7j  days 
Death  in  i3i  days 
Death  in  6^  days 

teenth  day,  whereas  animals  which  live  so  long  ordinarily  recover. 
Guinea-pig  32,  which  received  2.0  c.c,  showed  unusual  resistance. 
The  guinea-pigs  weighed  about  350  grams  each.  Other  experi- 
ments indicate  that  subcutaneous  and  intravascular  inoculations 
have  a  similar  degree  of  virulence. 

ACTIVE   IMMUNITY. 

I  have  not  learned  of  an  authenic  example  of  a  second  attack 
of  spotted  fever  in  man,  hence  it  has  seemed  somewhat  probable 
that  one  attack  renders  the  body  immune.  This  has  been  found 
to  be  true  in  a  recent  experiment  on  the  monkey. 

On  June  20,  1906,  Monkey  II  was  inoculated  with  the  blood  of 
Monkey  I,  the  latter  having  just  died  as  the  result  of  inoculation 
with  the  blood  of  the  patient,  Bradley.  Monkey  II  ran  a  typical 
course  of  fever,  showed  a  macular  eruption  of  the  perineal  skin 
on  the  eighth  day,  and  extensive  hemorrhages  of  the  scrotum, 
perineum,  back,  and  extremities  on  the  eleventh  day.  The  tem- 
perature returned  to  normal  on  the  twelfth  or  thirteenth  day, 
hence  the  eruption  became  hemorrhagic  as  convalescence  was 
about  to  begin.  The  animal  regained  its  former  health  and 
activity. 

Recently  this  animal  was  again  inoculated  with  the  blood  and 
tissues  of  an  infected  guinea-pig  (No.  36).  The  total  mass  of 
fluid  injected  was  8  c.c.  With  the  exception  of  some  indisposition 
on  the  following  day,  the  injection  produced  no  apparent  effect. 


296  Contributions  to  Medical  Science 

For  a  few  days  the  temperature  was  slightly  higher  than  before, 
but  during  this  time  and  since  the  monkey  has  shown  no  indis- 
position and  has  been  as  active  as  before  inoculation. 

By  way  of  control  another  monkey  of  the  same  size  (No.  IX) 
was  inoculated  with  the  same  amount  of  material,  the  same  quan- 
tity, and  by  the  same  route.  Between  the  third  and  fourth  days 
the  temperature  began  to  rise,  and,  until  it  was  killed  (7  days), 
it  ranged  between  105.  i  and  106.3.  The  conjunctivae  became 
reddened,  the  animal  shivered  at  frequent  intervals  and  sat,  inat- 
tentive and  with  head  drooping,  continuously.  On  the  fifth  day 
the  supra-orbital  skin  showed  reddened  patches,  and  on  the  sixth 
day  a  roseolar  eruption  was  visible  on  the  scrotum.  The  blood 
and  tissues  were  used  for  further  experiments. 

From  this  experiment  it  is  evident  that  one  attack  in  the  monkey 
confers  an  active  immunity,  and  on  the  basis  of  reasonable  analogies 
one  may  conclude  that  man  probably  acquires  a  similar  immunity. 

A  similar  acquired  immunity  has  also  been  demonstrated  in 
the  guinea-pig.  Its  duration,  beyond  the  period  of  two  months 
for  the  monkey  and  three  months  for  the  guinea-pig,  has  not  been 
determined. 

THE    LIFE   history   OF   DERMACENTOR   OCCIDENTALIS. 

On  account  of  the  suspicion  which  attaches  to  Dermacentor 
occidentalis  as  the  carrier,  or  a  carrier,  of  Rocky  Mountain  spotted 
fever,  its  Hfe  history  is  important.  The  following  brief  notes 
concerning  this  question  are  given  by  way  of  recording  the  changes 
which  were  observed  under  artificial  conditions.  No  description 
of  minute  morphologic  changes  will  be  given  at  this  time. 

May  30,  1906,  a  fully  developed  female  was  placed  in  a  cotton- 
stoppered  test-tube. 

June  21  (about)  the  tick  began  laying  eggs,  of  which  several 
hundred  (estimated)  were  eventually  deposited. 

June  28  or  29  hatching  began  and  continued  for  a  week  or  ten 
days,  resulting  in  six-legged  larvae  of  minute  size. 

July  13,  the  tube  with  its  contents  was  placed  in  a  tick-proof 
cage  with  a  guinea-pig.  The  larvae  took  hold  readily  for  the 
most  part,  but  did  not  feed  continuously.     They  increased  slightly 


Spotted  Fever  and  Dermacentor  Occidentalis       297 

in  size.  Eventually  all  remained  attached  to  the  skin  for  several 
successive  days,  grew  to  a  length  of  i  to  ij  mm.,  and  became 
swollen  and  of  a  uniform  dark  slate  color.  On  August  —  they  began 
to  drop  from  the  guinea-pig,  moved  to  a  safe  place,  and  went  into 
a  quiescent  state,  appearing  as  if  dead. 

August  5  they  began  to  molt,  throwing  off  a  snow-white  pellicle, 
and  came  out  as  eight-legged  nymphs.  After  7  to  10  days  the 
molting  of  the  brood  was  almost  complete.  When  they  first 
appeared  they  approximated  the  size  of  the  well-developed  larvae. 

When  again  placed  with  a  guinea-pig  or  monkey  they  seemed 
to  feed  intermittently  for  a  time,  but  eventually  attached  them- 
selves firmly  and  underwent  a  great  increase  in  size,  some  reaching 
a  length  of  about  3/16  of  an  inch.  With  the  increase  in  size,  the 
color  again  changed  to  that  of  a  uniform  gray-brown  or  slate  color. 
It  required  from  6  to  10  days  for  this  development  to  take  place 
after  the  ticks  were  placed  with  an  animal.  On  August  30  prac- 
tically all  the  ticks  which  remained  alive  had  undergone  this 
change. 

As  they  reached  the  condition  mentioned  they  were  removed 
and  placed  in  ventilated  boxes  where  they  went  almost  immediately 
into  a  second  quiescent  stage.  In  about  10  days  molting  again 
began,  a  white  mantle  being  thrown  off,  and  the  mites  emerged 
as  adult  ticks,  with  the  marking  which  one  finds  under  natural 
conditions  in  the  early  months  in  Montana. 

The  experiment  was  carried  on  entirely  at  room  temperature, 
and  the  various  stages  may  not  correspond  in  point  of  time  with 
those  taking  place  under  natural  conditions. 

Under  natural  conditions,  in  the  Bitter  Root  Valley,  the  adult 
ticks  begin  to  disappear  during  the  latter  part  of  June.  This 
probably  means  that  the  males,  and  those  females  which  have  not 
found  a  host,  die,  whereas  the  fully  developed  females  deposit 
their  eggs.  Locally,  knowledge  of  the  further  history  of  the  tick 
was  limited,  or  nil.  It  is  certain,  however,  that  during  the  warm 
summer  days,  the  conditions  are  favorable  for  the  hatching  of  the 
eggs,  and  for  the  development  of  the  larvae,  provided  a  host  is 
reached.  Inasmuch  as  a  crop  of  adult  ticks  is  not  observed  in  the 
fall,  it  is  possible  that  the  second  molting  stage,  mentioned  above, 


298  Contributions  to  Medical  Science 

represents  the  hibernating  stage  of  the  mite.  It  is  well  known, 
locally,  that  the  first  few  warm  days  of  the  spring  bring  out  the 
ticks.  These  few  warm  days  apparently  would  be  sufficient  to 
bring  to  completion  the  second  molting  stage.  On  the  other 
hand,  it  must  be  admitted  as  possible  that  the  second  molting  stage 
is  completed  in  the  late  fall,  and  that  hibernation  begins  at  once 
with  the  animal  fully  developed,  the  first  few  warm  days  of  spring 
being  sufficient  to  arouse  him. 

Observations  of  a  more  complete  nature  will  be  made  at  a 
future  date. 


OBSERVATIONS  ON  THE  VIRUS  AND  MEANS  OF  TRANS- 
MISSION OF  ROCKY  MOUNTAIN  SPOTTED  FEVER.' 

H.     T.     RiCKETTS. 

{From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

Investigations  into  the  nature  of  Rocky  Mountain  spotted  fever, 
its  etiology,  and  method  of  transmission,  have  been  carried  on  by 
the  writer  since  April,  1906. 

Previous  to  these  investigations  the  disease  had  been  studied 
extensively  by  Wilson  and  Chowning^  and  to  some  degree  also  by 
members  of  the  Pubhc  Health  and  Marine  Hospital  Service.  I 
shall  refer  to  these  investigations  only  to  say  that  Wilson  and 
Chowning  described  the  disease  as  a  pyroplasmosis,  and  advanced 
the  important  theory  that  man  is  infected  by  the  bite  of  the  ''wood- 
tick"  which  infests  the  mountainous  regions  of  Montana  and 
adjacent  Rocky  Mountain  states.  They  furnished  no  experimental 
proof  of  the  correctness  of  the  theory.  Following  the  report  of 
Wilson  and  Chowning,  Stiles,^  of  the  Public  Health  and  Marine 
Hospital  Service,  studied  the  disease  and  failed  utterly  to  find  the 
pyroplasma  of  the  former  investigators.  Stiles  discredited  also 
the  theory  of  transmission  by  the  tick,  but  without  experimental 
evidence  to  refute  the  theory. 

This  was  the  status  in  relation  to  the  etiology  and  means  of 
transmission  of  the  disease  when  my  studies  were  undertaken. 
The  results  which  I  have  obtained  have  been  described  briefly  in 
three  communications  to  the  Journal  of  the  American  Medical 
Association  (see  pp.  278,  288,  and  291  of  this  volume)."* 

The  essential  points  presented  in  these  articles  are  the  following : 
The  disease  was  transmitted  to  guinea-pigs  from  three  different 

■  Jour.  Infect.  Dis.,  1907,  4,  p.  141.  This  work  has  received  special  aid  from  the  Research  Fund  of 
the  American  Medical  Association,  from  Missoula  and  Ravalli  counties,  Montana,  from  the  Montana 
State  Board  of  Health,  from  the  University  of  Chicago,  and  the  Memorial  Institute  for  Infectious  Diseases. 

'Jour.  Am.  Med.  Assn.,  1902,  39,  p.  131;   also  Jour.  Infect.  Dis.,  1904,  r,  p.  31. 

>  Pub.  Health  and  Mar.  IIosp.  Bull.,  No.  20,  1903. 

*  (i)  "The  Study  of  'Rocky  Mountain  Spotted  Fever'  (Tick  Fever)  by  Means  of  Animal  Inocula- 
tions,"/o«r. /I  w.  A/eJ.  Assn.,  1906,  47,  p.  33;  (2)  "The  Transmission  of  Rocky  Mountain  Spotted 
Fever  by  the  Bite  of  the  Wood-Tick  (Dermacentor  occidentalis),"  ibid.,  1906,  47,  p.  358;  (3)  "Further 
Observations  on  Rocky  Mountain  Spotted  Fever  and  Dermacentor  occidentalis,"  ibid.,  1906,  47,  p.  1067. 

299 


300  Contributions  to  Medical  Science 

cases,  and  to  monkeys  from  two  different  cases  by  inoculation  with 
defibrinated  blood  from  the  patients  (i,  3).  From  the  last  of  the 
three  cases  the  disease  has  been  maintained  in  the  laboratory  by 
alternate  passage  through  the  monkey  and  the  guinea-pig,  by 
means  of  inoculations  with  blood  or  emulsions  of  organs  (3).  The 
first  attempts  to  maintain  the  infection  by  direct  inoculation  from 
guinea-pig  to  guinea-pig  failed,  possibly  for  reasons  which  will 
be  referred  to  below.  Rabbits  were  found  not  to  be  susceptible 
to  an  appreciable  degree,  and  the  same  was  true  of  white  mice  and 
white  rats.  In  a  preliminary  experiment  the  virus  did  not  pass 
through  a  Berkefeld  filter,  although  the  unfiltered  serum  was 
infectious.  The  anatomical  similarity  of  the  disease  produced 
in  the  guinea-pig  and  monkey  to  the  conditions  observed  in  man 
and  the  incubation  period  and  course  of  fever  induced  by  inocula- 
tion were  considered  sufficient  evidence  of  the  genuine  transmis- 
sion of  the  disease  to  these  animals  (1,3).  In  a  single  experiment 
a  tick,  female,  was  found  to  be  the  carrier  of  the  disease  from 
one  guinea-pig  to  another.  This  result  was  considered  as  tenta- 
tive (2).' 

The  virus  was  found  to  be  distributed  in  all  of  the  visceral 
organs,  the  most  vascular  organs  (liver,  spleen,  and  bone-marrow) 
and  the  kidney  apparently  being  the  most  infectious  (3). 

It  was  found  that  an  active  immunity,  which  possibly  is  relative 
in  degree,  is  established  in  the  monkey  and  guinea-pig  by  one 
attack  (3).  The  life-history  of  Dermacentor  occidentalis  was 
followed  under  laboratory  conditions.  This  tick  passes  through 
a  larval  stage  and  moults  twice  subsequently  before  reaching 
the  adult  form,  the  cycle  from  egg  to  adult  requiring  about  three 
months  under  the  modified  conditions,  which  prevailed  (3).  The 
tick  left  the  host  in  order  to  moult. 

It  is  the  purpose  of  this  paper  to  present  further  observations 
which  have  been  made  in  relation  to  the  distribution  of  the  virus 
in  the  body  fluids,  its  viability  and  resistance  under  different 
conditions,  its  filterability  and  certain  other  properties,  the  possi- 
bility of  transmission  by  means  of  the  bite  of  the  male  tick,  and 

■  King  also  has  reported  transmission  by  means  of  the  female  tick  (Pub.  Health  Reports,  July  37, 
1006). 


Means  of  Transmission  of  Spotted  Fever  301 

in  addition,  the  preservation  of  the  virus  by  uninterrupted  passage 
through  the  guinea-pig. 

the  distribution  of  the  virus  in  the  blood. 

Rocky  Mountain  spotted  fever  is  unquestionably  a  systemic  in- 
fection, since  the  disease  may  be  transmitted  by  the  inoculation 
with  blood  and  with  emulsions  of  the  solid  organs. 

A  study  of  the  distribution  of  the  virus  in  the  blood  was  under- 
taken with  the  hope  that  the  question  of  an  exclusive  or  predomi- 
nant erythrocytic  infection,  such  as  a  pyroplasmosis,  might  be 
affirmatively  or  negatively  determined.  Although  the  experi- 
ments reported  are  not  quantitative  in  character,  it  is  thought 
that  the  sum  total  of  results  renders  the  pyroplasma  theory  open 
to  suspicion.  Quantitative  work  is  very  difficult  from  the  fact 
that  the  concentration  of  the  virus  in  different  animals  is  subject 
to  considerable  variation;  this  may  be  as  great  as  a  tenfold  varia- 
tion, the  minimum  fatal  dose  of  defibrinated  blood  in  one  instance 
being  0.05  c.c.  and  in  another  0.5  c.c. 

The  following  results  have  been  obtained  in  regard  to  this 
phase  of  the  work: 

1.  It  is  impossible  to  free  the  blood  cells  of  defibrinated  blood 
from  the  virus  by  10  to  12  washings  with  physiologic  salt  solution 
(Table  3).  Repeated  washing,  however,  seems  to  decrease  the 
virulence  of  the  corpuscles  of  infected  blood  (Table  7). 

2.  As  washing  advances  a  point  is  reached  at  which  the  virus  is 
not  separated  from  the  corpuscles  in  infective  quantities  by  shaking 
with  salt  solution.  An  experiment  showed  that  a  fluid  of  the  loth 
washing  was  not  infectious  in  quantities  of  5  and  10  c.c,  whereas 
the  cellular  sediment  caused  the  disease  in  doses  of  0.7  and  1.5 
c.c.  (Tables  3  and  4). 

3.  The  serum  obtained  from  defibrinated  blood  or  from  spon- 
taneous clotting  is  infectious  in  doses  of  0.5  c.c.  or  even  less 
(Table  5). 

4.  Prolonged  centrifugation  of  the  serum  (6  hours)  does  not  free 
the  overlying  portion  from  the  virus  (Table  8). 

5.  An  exudate  of  leukocytes  in  the  pleural  or  peritoneal  cavity 
of  the  infected  monkey  or  guinea-pig,  caused  by  the  injection  of 


302 


Contributions  to  Medical  Science 


aleuronat  or  bouillon,  is  infectious  for  the  guinea-pig.  The  sedi- 
ment of  such  an  exudate  is  not  deprived  of  its  infectiousness  by 
repeated  washings,  and  the  overlying  fluid  remains  infectious  in 
spite  of  prolonged  centrifugation  (Table  6). 

The  following  selected  experiments  are  given  as  illustrative  of 
the  preceding  statements: 

TABLE  I. 
Control  Experiment.    Inocuiations  with  Fresh  Undefibrinated  Blood  of  Monkey  XIV. 


Guinea-Pig 
No. 

Dose 

Incubation  Period 

Duration  from  Date 
of  Inoculation 

Result 

Autopsy 

ISO 

0.05  c.c. 
0.1 
0.3 
0.7 

3-4  days 
3-4 
3-4 
3-4 

12    days 
loj 
II 
9 

Death 

Typical* 

" 

" 

•  The  following  conditions  at  autopsy  are  recognized  as  typical  for  spotted  fever  in  the  guinea-pig: 
Generalized  enlargement  of  the  lymph  glands  including  those  of  the  mesentery  and  mediastinum.  Either 
extreme  congestion  of,  or  hemorrhage  into,  the  lymph  glands.  Enlargement  of  the  spleen,  which  may 
reach  the  size  of  two  or  four  times  the  mass  of  the  normal  spleen.  The  spleen  is  much  congested  and 
cyanotic,  fairly  homogeneous  and  of  moderate  consistency,  i.e.,  neither  soft  nor  hard.  The  kidneys, 
suprarenal  glands,  and  liver  are  somewhat  enlarged  and  congested.  The  right  heart  and  veins  are  heavily 
engorged.  The  lungs  show  no  changes,  The  meninges  are  moderately  reddened.  The  bone-marrow  is 
rich  in  rather  dark  red  blood.  \"ery  frequent,  but  not  constant,  changes  are  hemorrhages  into  the  skin  of 
the  external  genitalia,  and,  in  males,  into  the  testicles  and  their  coverings.  Hemorrhage  also  occurs  with 
some  frequency  into  the  suprarenal  gland,  and  less  often,  into  the  liver.  If  visible  colonies  appear  on  a 
plate  of  the  heart's  blood  which  is  made  soon  after  death,  or  if  peritonitis  or  other  severe  local  inflammation 
is  found,  the  animal  has  been  disregarded. 


TABLE  2. 
Control  Experiment.    Inoculations  with  Fresh  Defibrinated  Blood  of  Monkey  XIV. 


Guinea-Pig 
No. 

Dose 

Incubation  Period 

Duration  from  Date 
of  Inoculation 

Result 

Autopsy 

154 

0.05  c.c. 
0.1 
0.3 
0.7 

S-6  days 
5-6 
3-4 
3-4 

13  days 
II 
12 
II 

Death 

Typical 

" 

156 

" 

" 

TABLE  3. 

Inoculations  with  Washed  Defibrinated  Blood  of  Monkey  XIV:  Ten  Washings,  the  Original 

Volume  Being  Restored. 


Guinea-Pig 
No. 

Dose 

Incubation  Period 

Duration  from  Date 
of  Inoculation 

Result 

Autopsy 

162* 

0.3  c.c. 

0.7 

I.O 

IS 

5  days 
S-6 

8  days 
13 

Not  sick 
KiUed 

Not  sick 
Death 

163 

Typical 

164 

165 :.. 

" 

Not  sick.    Died  later,  following  rectal  prolapse. 


Means  of  Transmission  of  Spotted  Fever  303 


TABLE  4- 
Inoculation  witb  Overlying  Salt  Solution  of  the  Tenth  Washing  of  Blood  Used  in  Table  3. 


No. 

Dose 

Result 

167 

lOC.C. 

Not  Sick 

TABLE  5- 
Inoculations  with  Serum  from  Blood  of  Monkey  XIV,  the  Serum  Being  Obtained  by  Defibrina- 
tion AND   CeNTRIFUGATION  FOR  OnE  HoUR. 


Guinea-Pig 
No. 

Dose 

Incubation  Period 

Duration  from  Date 
of  Inoculation 

Result 

Autopsy 

IS8 

159 

i6o* 

161 

1 .0 
2  .0 
30 
4.0 

S-4  days 
3-4 

3-4 

11  days 
13 

14 

Death 

Typical 

*  Accidental  death  in  4  days. 

TABLE  6. 
Infectious  Properties  of  Leukocytic  Exudate  from  Monkey  XIV,  Caused  by  an  Injection  of 

Aleuronat. 


Guinea- 
Pig  No. 

Dose  and  Material  Inoculated 

Incubation 
Period 

Duration  from 
Date  of  Inocula- 
tion 

Result             Autopsy 

t47 

148 

149 

2  c.c.  of  unaltered  exudate 
5  c.c.  of  leukocj^e  suspen- 
sion, washed  3  times 
4J  c.c.   of  fluid  from  first 
centrifugation 

6  days 
6 

&-7 

13  days 

14 

21 

Death             Typical 
Recovery 

TABLE  7. 

The  Effect  of  Repeated  Washing  of  Blood  in  Decreasing  Its  Virulence.    Mixed  Bloods  of 

Monkeys  VII  and  VIII,  12  c.c.  Injected  into  Each  Guinea-Pig. 


Guinea-Pig 

No. 

Washings 

Incubation  Period 

Duration  from  Date 
of  Inoculation 

Result 

Autopsy 

44 

I 

2 

S 

10 

2  days 

4 

5 
5 

7  days 
10 
14 
16 

Death 

Typical 

ta::::::::::: 

47 

'• 

TABLE  8. 

Attempt  to  Remove  Infectious  Properties  of  Serum  by  Centrifugation  for  Six  Hours  at  the 

Rate  of  2,000  Revolutions  per  Minute,    s  c.c.  of  Serum  from  Monkeys  VII  and  VIII. 


Guinea-Pig 

No. 


Injection 


50 Centrifugation  serum 

SI Final  o.  5  c.c.  of  above 


Incubation 
Period 


7-8  days 
S 


Duration 


20  days 
16 


Result 


Recovery 


304  Contributions  to  Medical  Science 

The  tenacity  with  which  the  virus  associates  itself  with  the 
blood  corpuscles  suggests  that  a  certain  proportion  of  the  parasites 
may  be  within  either  the  leukocytes  or  the  erythrocytes.  This 
suggestion  seems  the  stronger  since,  as  shown  in  guinea-pig  167 
(Table  4),  a  certain  proportion  of  the  virus  is  not  readily  separated 
from  the  corpuscles  by  moderate  agitation  with  salt  solution,  but 
remains  rather  firmly  associated  with  the  blood  cells  (Table  3). 
In  relation  to  this  fact,  however,  the  following  points  are  to  be 
observed:  First,  the  density  and  viscosity  of  serum  is  consider- 
ably greater  than  that  of  0.85  per  cent  salt  solution,  and  although 
the  virus  cannot  be  separated  from  serum  readily  by  centrifuga- 
tion,  such  separation  could  be  more  readily  accomplished  in  salt 
solution.  Hence  it  is  still  possible  that  the  absence  of  the  virus 
in  pathogenic  quantity  from  the  salt  solution  of  the  loth  washing 
may  be  caused  in  large  part  by  the  sedimentation  necessary  for 
the  separation  of  the  corpuscles.  Not  only  must  the  difference 
in  the  density  of  serum  and  salt  solution  cause  a  wide  difference 
in  the  ease  with  which  microbes  may  be  sedimented  from  their 
solutions,  but  the  coarse  physical  structure  of  serum  must  interfere 
greatly  with  the  sedimentation  of  minute  particles.  In  the  second 
place  it  is  readily  conceivable  that  minute  organisms  may  adhere 
to  the  external  surface  of  erythrocytes  and  leukocytes  so  firmly 
that  moderate  a'gitation  in  salt  solution  does  not  cause  their 
separation  in  pathogenic  quantities. 

In  an  experiment,  the  protocol  of  which  is  not  given,  o.  5  c.c.  of 
serum  obtained  after  spontaneous  clotting  caused  fatal  infection 
in  the  guinea-pig.  This  would  suggest  a  general  plasmatic  infec- 
tion rather  than  one  which  is  essentially  cellular. 

Since  a  leukocytic  exudate  practically  devoid  of  erythrocytes 
is  infectious,  it  seems  probable  that  the  condition  could  not  well 
be  considered  as  an  essential  involvement  of  the  erythrocytes. 
Furthermore,  from  the  fact  that  the  virxis  exists  in  the  fluid 
portion  of  the  leukocytic  exudate  in  pathogenic  quantities,  the 
infection  could  hardly  be  considered  as  essentially  leukocytic  in 
character. 

Although  it  is  desirable  to  wait  until  certain  quantitative  experi- 
ments are  completed  before  drawing  positive  conclusions  as  to  the 


Means  of  Transmission  of  Spotted  Fever  305 

situation  of  the  virus,  I  believe  the  qualitative  results  described 
above  indicate  a  general  plasmatic  infection  rather  than  an  essen- 
tial invasion  of  blood  cells. 

FILTRATION  AND   INTOXICATION. 

Filtration  experiments  have  been  continued  with  variations 
in  conditions.  Small  Berkefeld  candles  have  been  used  exclu- 
sively and  for  the  most  part  those  which  were  fresh  from  the 
factory.  In  case  a  filter  had  been  previously  used,  it  was  washed 
with  distilled  water  in  both  directions,  subjected  to  prolonged 
boiling  in  a  solution  of  sodium  carbonate,  and  washed  first  with 
salt  solution  and  then  with  distilled  water  in  both  directions, 
before  being  used  again.  It  has  seemed  useless  to  employ  porce- 
lain filters  so  long  as  the  virus  has  not  been  passed  through  the 
more  porous  Berkefeld  filters. 

No  greater  pressure  has  been  used  than  that  obtained  from  a 
vacuum  water  pump,  with  a  pressure  of  from  30  to  40  pounds  in 
the  mains.  In  all  experiments,  except  one,  the  filters  have  been 
kept  covered  with  the  serum  by  drawing  the  latter  into  a  pipette, 
then  letting  it  fall  over  the  surface  of  the  filter.  In  the  exception 
mentioned,  the  filter  was  covered  with  a  rubber  tube  which  extended 
one-half  to  three-quarters  of  an  inch  above  the  height  of  the  filter, 
the  serum  being  fed  into  the  cup  made  by  the  projection  of  the 
tube.  In  this  particular  instance  the  attempt  was  made  to  filter 
the  serum  undiluted.  After  a  time,  however,  the  filtration  pro- 
ceeded so  slowly  that  salt  solution  was  added.  In  the  remaining 
experiments  an  equal  quantity  of  salt  solution  was  added  to  the 
serum  before  filtration  was  begun,  and  in  all  cases  the  filter  has 
been  washed  out  with  several  cubic  centimeters  of  salt  solution 
after  the  serum  had  passed  through.  The  serum  of  infected 
monkeys  has  been  used  throughout  for  filtration  experiments, 
the  serum  being  obtained  by  defibrination  and  centrifugation. 
In  all  instances  the  infectiousness  of  the  unfiltered  serum  has  been 
determined  by  control  experiments  and  the  serum  has  been  used 
as  soon  as  possible  after  obtaining  it. 

In  no  instance  has  infected  serum  even  in  quantities  of  6  to 
12  c.c.  been  infectious  for  the  guinea-pig  after  being  filtered  in  the 


3o6  Contributions  to  Medical  Science 

manner  described,  although  0.5  c.c.  of  fresh  defibrinated  blood 
has  not  failed  to  cause  the  disease  (see  Table  9,  as  an  illustration). 

TABLE  g. 

Filtration  Experiment.    Serum  of  Monkey  XII.    5  c.c.  of  the  Diluted  Serum  Were  Injected 

Intraperitoneally  and  the  Remaining  Portion  Subcutaneously. 


Guinea-Pig 
No. 

V'olume  of  Serum 

''tZ:Jr       i  Incubation  Period 

Result 

Autopsy 

Filtered 

Unfiltered 

7  c.c. 

7 

g-io  c.c. 
9-10 

7  days 

Not  sick 

Death          TjT)ical 

In  view  of  the  possibility  that  minute  forms  of  the  organism, 
capable  of  passing  through  the  Berkefeld  filter,  might  exist  within 
the  erythrocytes  or  leukocytes,  whereas  only  larger  forms  might 
be  extracellular,  an  attempt  was  made  to  free  such  hypothetical 
minute  forms  by  crushing  the  blood  cells  in  a  porcelain  ball-mill. 
Inasmuch  as  the  result  of  this  experiment  points  to  the  existence 
of  a  toxic  substance  in  infected  blood,  the  details  may  be  given. 

Defibrinated  blood  from  monkey  No.  XIV  was  washed  lo  times 
with  sterile  physiologic  salt  solution  in  order  to  get  rid  of  the  serum. 
This  washed  blood  proved  to  be  infectious  for  guinea-pigs  in  doses 
of  o.  7  and  i .  5  c.c.  (see  Table  3). 

The  washed  cells  from  25.2  c.c.  of  blood  were  ground  in  the 
ball-mill  for  six  hours.  The  mass  was  removed  from  the  mill 
by  fractional  washing  with  salt  solution,  centrifugated  to  get 
rid  of  porcelain  sand,  the  latter  then  being  washed  fractionally, 
and  the  total  volume  of  fluid  being  made  up  to  50.4  c.c.  by  addi- 
tional salt  solution.  The  fluid  was  dark  red  in  color,  cloudy,  and 
no  cells  could  be  recognized  by  microscopic  examination.  Some 
of  this  fluid,  representing  50  per  cent  of  blood  in  volume,  was 
injected  into  guinea-pigs  in  doses  of  1.4,  2.0,  5.0,  and  10. o  c.c, 
the  effects  of  which  are  shown  in  Table  10. 

The  remaining  portion  was  passed  through  a  fresh  Berkefeld 
filter  about  three  hours  being  occupied  in  filtration.  Filtration 
proceeded  rapidly  at  first,  but  more  slowly  later,  as  the  fluid 
became  more  concentrated  in  insoluble  albuminous  particles. 
The  filtrate  was  dark  red  and  perfectly  clear.  The  entire  amount 
was  injected  intraperitoneally  into  two  guinea-pigs  at  interrupted 
periods  (Table  11). 


Means  of  Transmission  of  Spotted  Fever 


307 


In  comparing  Tables  3,  10,  and  11,  which  represent  experiments 
performed  with  the  same  blood,  one  gains  the  impression,  first, 
that  grinding  the  blood  in  the  mill  had  almost  entirely  destroyed 
its  infectiousness,  and  second,  that  with  the  destruction  of  the 
infectiousness  of  the  blood  the  latter  manifested  a  rather  pro- 
nounced toxicity. 

TABLE  10. 

Test  of  the  Infectiousness  of  the  Washed  and  Ground-up  Corpuscles  of  Monkey  XiV. 


Guinea- 
Pig 
No. 

Volume  Injected 

Equivalent  in  Nor- 
mal Blood  Volume 
Less  Estimated 
10%  Loss 

Result 

170 

171 

>72 

173 

I  .4  c.c. 

2.0 

50 

10. 0 

0.63  c.c. 

0.9 

2.2s 

4S 

Slight  febrile  reaction  beginning  on  the  second  day  after 

inoculation.     Recovery 
Moderate  febrile  reaction  beginning  the  first  day  after 

inoculation.     Recovery 
Moderate  febrile  reaction  beginning  the  first  day  after 

inoculation.     The  primary  temperature  subsided,  but 

in  6  days  a  course  of  fever  developed  which  persisted 

for  9  days.     Recovery 
Similar  to  that  of  172,  but  died  on  the  27th  day  after 

inoculation.    Death  due  to  extraneous  infection,  as 

shown  by  culture 

In  support  of  the  first  point,  it  may  be  noted  in  Table  3  that  the 
blood  before  it  was  ground  up  produced  typical  infections  in  doses 
of  0.7  c.c.  and  1.5  c.c,  whereas  after  it  was  ground  up,  its  infec- 
tiousness had  been  largely  destroyed,  as  seen  in  Table  10.  This 
effect,  possibly,  is  due  to  an  actual  crushing  of  the  organism  in  the 
mill. 

TABLE  II. 
Showing  the  Toxic  Effects  of  the  Filtrate  of  Washed  and  Ground-up  Corpuscles  from  the 

Blood  of  Monkey  XIV. 


Guinea- 
Pig 
No. 

Volume  Injected 
Fractionally 

Equivalent  in  Nor- 
mal Blood  Volume 
Less  Estimated 
15%  Loss 

Result 

174 

J7S 

IS  c.c. 
20 

6.4  c.c. 
8.5 

Death   in   4   days   following   febrile   reaction.     Blood 

sterile 
Death   in   6   days   following   febrile   reaction.     Blood 

sterile 

In  support  of  the  second  point,  we  have  to  note  first  the  primary 
fever  which  developed  in  the  guinea-pigs  of  Table  lo,  and  second, 
the  toxicity  of  the  filtrate  as  shown  in  Table  ii.  In  order  to 
determine  whether  the  intoxication  seen  in  the  animals  of  Table 
II  was  due  to  microbic  poison  or  to  the  action  of  the  proteids  of 


3o8  Contributions  to  Medical  Science 

monkey  corpuscles  on  the  guinea-pig,  12  c.c.  of  normal  monkey 
blood  were  ground  up  and  a  filtrate  prepared  analogous  to  that 
injected  into  guinea-pigs  Nos.  174  and  175.  The  total  filtrate, 
20  c.c,  was  injected  into  a  single  guinea-pig  which  had  the  weight 
of  the  former  animals.  A  shght  rise  of  temperature  which  occurred 
on  the  fifth  day  after  injection,  lasted  two  days;  otherwise  there 
was  no  disturbance.  It  may  accordingly  be  concluded  that  the 
intoxication  of  animals  Nos.  174  and  175  was  not  due  to  the  normal 
proteids  of  the  corpuscles  of  monkey's  blood.  One  could  scarcely 
consider  the  condition  an  infection,  since  the  same  blood  before 
filtration  had  shown  practically  no  infectious  properties  (Table  10). 
It  could  hardly  be  due  to  soluble  toxins  in  the  monkey's  blood, 
since  such  toxins  probably  would  have  been  removed  by  the 
washings  to  which  the  blood  had  been  subjected.  Although 
further  experiments  are  indicated  before  positive  conclusions 
are  drawn,  it  seems  probable  that  the  intoxication  of  the  animals 
was  due  to  the  liberation  of  poisons  by  the  crushing  of  the  virus  in 
the  ball-mill. 

resistance  to  heat. 

Heating  experiments  have  been  performed  at  temperatures  of 
45°  and  50°  C.  It  was  necessary  to  use  rather  large  quantities  of 
blood  in  order  to  be  certain  that  a  fatal  quantity  of  organisms  was 
being  dealt  with.  On  this  account  it  was  preferable  to  use  rela- 
tively low  temperatures  over  rather  long  exposures  in  order  to 
insure  as  complete  diffusion  of  the  heat  as  possible. 

Results. — In  an  experiment  in  which  3 . 5  c.c.  of  blood  were 
heated  at  45°  C.  for  5,  10,  15,  20,  25,  and  30  minutes,  all  animals 
died  of  spotted  fever.  In  two  experiments  in  which  the  blood  was 
heated  at  50°  C,  the  infectiousness  of  the  virus  was  destroyed  in 
25  minutes  in  one  series  and  in  30  minutes  in  another. 

RESISTANCE   TO  DESICCATION. 

The  following  is  the  technic  used  in  desiccation  experiments: 
Uniform  quantities  of  blood  are  distributed  into  open  Petri  dishes 
which  are  placed  in  a  desiccator  over  sulphuric  acid  and  dried  as 
quickly  as  possible  under  vacuum  at  room  temperature.     Desicca- 


Means  of  Transmission  of  Spotted  Fever  309 

tion  requires  from  18  to  24  hours,  depending  on  the  degree  of 
exhaustion  of  the  bell-jar.  When  desiccation  is  complete  the  plates 
are  placed  in  an  ordinary  sulphuric-acid  desiccator  in  the  ice-chest. 
Results. — Two  series  of  experiments  showed  the  loss  of  patho- 
genicity at  some  time  between  24  and  48  hours  after  complete 
desiccation. 

VIABILITY   IN   THE   ICE-CHEST. 

The  M.L.D.  of  the  blood  of  monkey  No.  XV,  which  when 
drawn  was  about  o.i  c.c,  had  increased  noticeably  in  five  days, 
reached  2.0  c.c.  in  11  days,  and  in  15  days  3.0  c.c.  failed  to  produce 
infection.  The  blood  was  kept  in  the  ice  compartment  of  the  ice- 
chest.  In  another  instance  5.0  c.c.  retained  infectiousness  for 
16  days. 

transmission   by   means   of   the   MALE   TICK. 

The  possibility  of  transmission  by  means  of  the  male  tick 
(Dermacentor  occidentalis)  has  been  demonstrated  conclusively  in 
a  recent  experiment.  The  tick  was  one  which  had  been  raised  from 
the  egg  in  the  laboratory,  the  life-history  of  the  brood  having  been 
published  previously  {loc.  cit.). 

Infection  of  the  tick  was  accomplished  by  feeding  on  two  sick 
guinea-pigs  in  the  following  way:  On  October  16  it  was  placed  on 
the  ear  of  guinea-pig  No.  107,  where  it  remained  for  about  12  hours, 
at  the  end  of  which  time  the  guinea-pig  died.  Two  days  later  it 
was  placed  on  the  ear  of  guinea-pig  No.  121,  where  it  remained  for 
about  20  hours,  or  until  the  guinea-pig  died.  After  an  intermission 
of  three  days  the  tick  was  placed  on  the  ear  of  the  healthy  guinea- 
pig  (No.  169),  and  the  latter  died  in  13  days,  showing  changes  which 
have  been  recognized  as  characteristic  of  spotted  fever.  However, 
since  an  adventitious  epidemic  had  developed  among  the  guinea-pigs 
and  since  areas  of  focal  necrosis  found  in  the  spleen  of  guinea-pig 
No.  169  were  not  entirely  typical  for  spotted  fever  in  the  guinea- 
pig,  the  experiment  was  not  considered  conclusive  and  the  animal 
was  discarded.  On  November  7,  17  days  after  the  tick  had  been 
removed  from  the  infected  guinea-pig  (No.  121),  it  was  again 
placed  on  a  healthy  guinea-pig  (No.  182).  It  was  allowed  to  remain 
attached  for  3^  days,  after  which  it  was  removed.     Ten  days 


3IO  Contributions  to  Medical  Science 

after  the  tick  was  placed  on  the  guinea-pig  the  latter  suddenly 
developed  high  fever  and  died  in  five  days,  showing  those  anatomi- 
cal changes  which  have  proved  to  be  diagnostic  of  experimental 
spotted  fever. 

Autopsy. — Scrotum  moderately  hemorrhagic  and  very  cyanotic.  Hemorrhagic 
condition  is  seen  best  by  the  naked  eye  in  cutting  through  the  skin.  Testicular 
coverings  are  moderately  congested  and  the  anterior  pole  of  the  testicles  deeply 
infiltrated  with  blood.  The  axillary,  inguinal,  and  mesenteric  lymph  glands  are 
enlarged  and  hemorrhagic.  The  spleen  is  several  times  the  mass  of  the  normal  spleen 
and  cyanotic  in  color.  The  kidneys  are  congested  and  cyanotic;  suprarenal  glands  are 
enlarged;  liver  enlarged,  congested,  and  cyanotic.  The  lungs  and  heart  show  no 
appreciable  changes.  The  meninges  are  slightly  reddened.  At  the  point  of  the  tick 
bite  is  a  necrotic  crusted  wound  about  one-fourth  inch  in  diameter.  Cultures  from 
the  heart  and  peritoneum  yielded  no  growth. 

From  the  organs  of  this  guinea-pig  inoculations  were  made  into 
two  other  guinea-pigs,  one  of  which  died  in  seven  and  the  other  in 
eight  days,  both  showing  typical  anatomical  and  clinical  phenomena 
of  spotted  fever.  Monkey  No.  XVII  which  was  also  inoculated  from 
guinea-pig  No.  182  ran  a  typical  course  and  presented  extensive 
scrotal  hemorrhage. 

From  the  second  generation  in  guinea-pigs  and  also  from  the 
monkey,  inoculations  were  made  into  a  third  generation,  the 
members  of  which  ran  typical  courses.  This  is  being  continued  by 
successive  inoculations  of  the  guinea-pig. 

The  experiment  is  regarded  as  conclusive. 

continuous  passage  through  the  guinea-pig. 

In  a  previous  article  {loc.  cit.)  my  failure  to  keep  spotted  fever 
alive  by  the  successive  inoculation  of  guinea-pigs  was  referred  to. 
In  the  earlier  attempts  fresh  inoculations  were  made  only  as  the 
guinea-pigs  were  about  to  die  or  after  they  had  died.  The  possi- 
bility was  recognized  that  the  quantity  of  living  virus  in  an  infected 
animal  may  be  greater  early  in  the  course  of  the  disease  than  at 
the  time  of  death,  hence  at  a  convenient  time  the  attempt  was 
made  to  perpetuate  the  infection  in  the  guinea-pig  alone  by  inocula- 
tion with  blood  or  organs  taken  on  the  third  to  the  fifth  day  after 
fever  had  begun.  This  method  has  proved  entirely  successful 
through  five  and  into  six  generations  of  guinea-pigs.     Hence  it 


Means  of  Transmission  of  Spotted  Fever  311 

seems  probable  that  the  monkey  can  be  dispensed  with  for  the 
purpose  of  maintaining  the  disease  in  the  laboratory. 

SUMMARY. 

Rocky  Mountain  spotted  fever  is  transmissible  to  the  guinea- 
pig  and  monkey  by  the  inoculation  of  defibrinated  blood  of  patients 
suffering  from  the  disease. 

The  virus  may  be  kept  ahve  in  the  laboratory  either  by  alternate 
inoculation  of  monkey  and  guinea-pig,  or  by  continuous  passage 
through  the  guinea-pig  by  observing  the  method  described  above. 

The  disease  is  transmissible  from  one  animal  to  another  by 
means  of  the  bite  of  either  the  male  or  female  tick  (Dermacentor 
occidentalis) . 

One  attack  of  the  disease  establishes  a  rather  high  degree  of 
immunity  to  subsequent  inoculation. 

Attempts  to  pass  the  virus  through  Berkefeld  filters  have  failed. 

The  parasites  are  not  located  essentially  in  either  erythrocytes 
or  leukocytes  but  are  present  in  the  body  fluids  generally. 

By  grinding  infected  blood  in  the  ball-mill  infectiousness  is 
largely  destroyed ;  in  this  process  there  is  some  reason  to  think  that 
the  organisms  are  crushed  and  that  toxic  substances  are  thereby 
liberated. 


THE  ROLE  OF  THE  WOOD-TICK  (DERMACENTOR 
OCCIDENTALIS)  IN  ROCKY  MOUNTAIN  SPOTTED 
FEVER,  AND  THE  SUSCEPTIBILITY  OF  LOCAL  ANI- 
MALS  TO  THIS  DISEASE/ 

A   PRELIMINARY  REPORT. 

H.     T.     RiCKETTS. 
(From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

THE   TECHNIC   OF   FEEDING   TICKS   ON   THE   GUINEA-PIG. 

Many  embarrassments  have  been  encountered  in  experiments 
in  which  the  tick  is  allowed  to  feed  on  the  guinea-pig.  There 
are  few  spots  on  the  body  of  the  latter  which  he  cannot  reach  with 
his  teeth  or  with  the  toes  of  his  hind  legs,  and,  since  the  bite  of 
the  tick  is  very  irritating,  ticks  are  frequently  dislodged  or  killed, 
provided  the  guinea-pig  is  not  restrained  properly.  The  tick  is 
prone  to  attach  itself  to  the  delicate  hairless  skin  behind  the  ear 
or  to  the  ventral  surface  of  the  body  in  the  vicinity  of  the  mammary 
glands.  When  in  the  former  position  it  is  readily  dislodged  by 
the  nails  of  the  pig's  hind  feet,  and  when  in  the  latter  position 
may  be  killed  by  biting.  In  order  to  obviate  some  of  these  acci- 
dents and  to  prevent  the  loss  of  valuable  ticks,  the  following 
fairly  satisfactory  technic  has  been  devised: 

When  testing  or  feeding  an  individual  tick,  the  guinea-pig  is  placed  in  a  pillory 
for  a  short  time  and  the  tick  is  allowed  to  attach  itself  to  the  ear  or  to  the  back  of  the 
head  or  neck.  The  pillory  prevents  the  pig  from  scratching  the  head,  and  because 
of  the  temporary  confinement  he  seems  not  to  notice  the  presence  of  the  tick  until  the 
latter  is  more  or  less  firmly  attached.  Even  under  these  conditions,  however,  the 
process  is  often  tedious,  since  the  tick,  before  it  is  firmly  attached,  may  be  thrown  off 
by  a  violent  shake  of  the  guinea-pig's  head.  After  the  tick  appears  to  be  attached, 
usually  within  ten  to  twenty  minutes,  the  pillory  with  the  pig  in  place  is  tied  up  in 
a  sack  having  the  bottom  and  sides  of  canvas  and  the  top  of  cheesecloth,  this  condition 
being  maintained  for  one  or  two  hours. 

At  the  end  of  this  time  the  animal  is  removed  from  the  pillory  and  placed  in  a 
skeleton  cage  which  is  surrounded  by  a  sack  like  that  described  above,  a  collar  of  light 
linoleum  first  having  been  placed  about  the  neck  of  the  pig.  The  collar  is  of  such 
width  (i  to  i^  inches)  that  it  precludes  scratching  the  head  parts,  and  also  prevents  the 
biting  of  ticks  which  have  become  attached  to  the  ventral  skin.  This  width  also 
permits  the  collar  to  rest  on  the  bottom  of  the  cage,  thus  removing  the  weight,  which  is 
sHght,  from  the  neck  of  the  guinea-pig. 

'From  Jour.  Am.  Med.  Assn.,  1907,  49,  p.  24. 

312 


Role  of  the  Wood-Tick  in  Spotted  Fever  313 

The  cage  is  made  of  a  frame  of  wood  slats,  and  has  a  floor  of  ^-inch  mesh  wire, 
which  is  attached  to  the  frame  an  inch  above  its  bottom.  This  wire  floor  permits 
the  escape  of  water  and  grain  to  the  dead  space  below,  and  also  gives  free  ticks  refuge 
from  the  teeth  of  the  guinea-pig,  the  mesh  being  suflSciently  large  to  permit  ticks  of 
ordinary  size  to  pass  below. 

When  it  is  desired  to  feed  a  large  number  of  ticks,  the  guinea-pig,  with  its  collar 
in  place,  is  confined  to  the  cage  with  the  ticks  at  large,  the  latter  being  allowed  to  feed 
at  will.  Under  this  method  large  numbers  of  ticks  can  be  fed  with  a  minimal  loss 
and  a  small  expenditure  of  time. 

Infection  of  the  Nymph. 

Among  a  group  of  ticks  received  from  Mr.  F.  D.  Nichols  of  Hamilton,  Mont., 
January  i,  1907,  were  four  nymphs.  They  had  been  taken  from  horses  a  few  days 
previously,  south  of  Hamilton,  in  a  district  not  known  to  be  infected  with  spotted 
fever.     One  of  the  nymphs  was  moribund  at  the  time  they  were  received. 

January  3,  at  5  p.m.,  the  three  remaining  nymphs  were  placed  on  an  infected 
guinea-pig  (290)  from  which  they  were  removed  three  days  later.  Guinea-pig  290 
represented  the  eleventh  consecutive  guinea-pig  passage  of  the  strain  which  was 
obtained  in  the  spring  of  1906,  the  custom  of  infecting  the  monkey  and  guinea-pig 
alternately  having  been  abandoned  some  time  previously.  Guinea-pig  290  ran  a 
perfectly  tjTpical  course  clinically,  the  anatomic  changes  were  characteristic,  and 
cultures  from  the  heart  and  organs  remained  free  from  bacterial  growth. 

It  was  the  intention  at  this  time  to  place  the  nymphs  individually  on  healthy 
guinea-pigs  in  order  to  test  their  infectivity.  They  entered  the  moulting  stage, 
however,  almost  at  once,  and  it  was  impossible  to  make  this  test. 

Nymph  i  came  out  of  its  moult  January  27,  twenty-one  days  having  been  occu- 
pied in  the  process;  whereas  the  moulting  period  of  nymph  2  lasted  thirty-four  days. 
Nymph  3  died  during  the  moulting  period.     Nymphs  i  and  2  appeared  as  females. 

The  further  history  of  tick  i  is  as  follows:  January  29  it  would  not  feed.  On 
February  2  it  attached  itself  to  the  ear  of  guinea-pig  332,  from  which  it  was  removed 
after  fifty-seven  hours.     It  had  increased  rather  markedly  in  size  during  the  feeding. 

Temperature  of  Guinea-Pig  332. 

February  4 102 . 6  F.  February  7 104  F. 

February  5 103       F.  February  8 106  F. 

February  6 102.2  F.  February  9 105  F. 

February  10 Had  been  dead  for  some  hours. 

Autopsy. — Enlarged  and  hemorrhagic  axillary,  inguinal,  and  mesenteric  lymph 
glands.  Spleen  was  several  times  the  normal  size  and  of  a  deep  bluish-red  color, 
and  rather  coarsely  granular.  The  suprarenal  glands  were  much  enlarged  and  con- 
gested. The  kidneys  and  the  liver  were  congested,  but  did  not  appear  degenerated. 
The  abdominal  viscera  showed  postmortem  discoloration.  The  heart,  lungs,  and 
intestines  were  normal.  The  skin  of  the  external  genitals  was  not  congested  nor 
hemorrhagic,  and  no  eruption  was  discernible  on  the  skin.  A  plate  culture  from 
the  heart's  blood  gave  no  bacterial  growth. 

Diagnosis. — Spotted  fever. 

The  short  incubation  period,  the  sudden  and  high  rise  in  the  temperature  of  the 
animal  and  its  early  death  are  features  which  indicated  a  highly  virulent  infection. 
Under  these  conditions  experience  has  shown  that  the  skin  manifestations  do  not 
always  develop. 


314  Contributions  to  Medical  Science 

On  account  of  postmortem  changes  in  the  viscera,  transfer  of  the  infection  from 
the  dead  animal  was  not  attempted. 

Second  transmission. — On  February  g,  after  an  interval  of  five  days,  the  tick 
became  attached  to  guinea-pig  363,  from  which  it  was  removed  after  feeding  for 
twenty-two  and  one-half  hours. 

Temperatitre  of  Guinea-Pig  363. 

February  11 103 . 4  F.  February  13 105 . 4  F. 

February  12 103.8  F.  February  14 105.5  F. 

February  15 105 . 2  F.     Blood  taken  from  the  heart. 

February  16 104      F.  February  17 103.9  F. 

February  18 103.8  F.    Scrotum  swollen  and  congested. 

February  19 Dead. 

Autopsy  (immediately  after  death). — Lymph   glands  were  moderately  enlarged 
and  congested.    No  changes  were  found  in  the  liver,  lungs,  heart,  or  intestines.     There 
were  no  cutaneous  hemorrhages,  but  the  skin  of  the  face  and  external  genitals  was 
extremely  cyanotic.     A  plate  culture  of  the  heart's  blood  remained  sterile. 
Diagnosis. — Spotted  fever. 

The  3  c.c.  of  blood  drawn  from  the  heart  February  15  were  injected  intraperi- 
toneally  into  guinea-pig  376  immediately.     The  following  course  of  fever  was  recorded: 

February  16 103 . 4  F.  February  19 105       F. 

February  17 103. i  F.  February  20 106.2  F. 

February  18 105       F.  February  21 105       F. 

February  22 96. 2    F.     Killed  by  chloroform  when  moribund. 

The  findings  at  autopsy  were  identical  with  those  recorded  above  and  need  not 
be  repeated.  Plate  cultures  of  the  heart's  blood,  spleen,  and  liver  remained  sterile. 
In  making  the  plate  cultures  of  the  organs  fragments  of  the  tissues  were  triturated 
in  bouillon  and  the  emulsion  thus  obtained  was  mixed  with  the  liquefied  agar  before 
plating. 

Third  transmission. — On  February  26  the  tick  attached  itself  to  the  ear  of  guinea- 
pig  406,  where  it  was  allowed  to  remain  for  thirty  hours.  On  March  3  the  temperature 
rose  to  104. 1  F.,  in  the  vicinity  of  which  mark  it  remained  until  March  6,  when  the 
animal  died.  The  anatomic  findings  were  as  described  above,  and  cultures  from  the 
heart,  liver,  and  spleen  were  likewise  sterile.  The  skin  of  the  scrotum  was  deeply 
congested. 

The  second  nymph  underwent  a  positive  test  on  guinea-pig  364  on  February  10, 
after  a  feeding  period  of  twenty-two  and  one-half  hours.  Blood  was  drawn  from  the 
heart  of  this  animal  on  the  first  day  of  fever  (February  15)  and  3  c.c.  were  inoculated 
into  guinea-pig  377.  The  latter  animal  ran  a  typical  course  and  was  killed  on  the 
sixth  day  after  infection  in  order  to  obtain  material  for  the  inoculation  of  monkey  26. 
The  anatomic  changes  were  characteristic  and  the  bacteriologic  result  was,  as  usual, 
negative. 

Ten  cubic  centimeters  of  defibrinated  blood  from  guinea-pig  377  were  given  to 
monkey  26  intraperitoneally  (February  21).    The  following  course  of  fever  developed: 

February  22 103       F.  February  25 105 . 3  F. 

February  23 105 . 6  F.  February  26 105       F. 

February  24 105       F.  February  27 104.4  F. 

Killed  February  27,  the  blood  being  utilized  for  various  experiments. 
The  anatomic  changes  were  tj^pical  and  cultures  from  the  blood  and  viscera  gave 
no  growth. 


Role  of  the  Wood-Tick  in  Spotted  Fever  315 

In  order  to  test  the  validity  of  the  experiment  still  further  three  guinea-pigs  were 
inoculated  with  the  blood  of  monkey  26.  All  three  developed  spotted  fever,  exhibit- 
ing typical  symptoms  and  anatomic  changes. 

These  results  are  of  especial  importance  in  two  particulars:  In 
the  first  place  it  must  be  considered  that  the  tick  in  its  role  as  the 
carrier  of  spotted  fever  is  itself  the  victim  of  an  infection;  that 
the  transmission  is  not  purely  mechanical.  The  following  facts 
support  this  conclusion:  First,  the  tick  called  nymph  i  retained 
its  infectivity  for  fifty-four  or  fifty-five  days,  having  in  the  mean- 
time passed  through  the  moulting  stage,  whereas  outside  the 
living  body  the  viabiUty  of  the  virus  under  the  most  favorable 
conditions,  i.e.,  in  the  ice-chest  and  in  the  dark,  is  less  than  half 
this  period  and  may  be  only  one-third  as  long. 

If  we  assume  that  the  virus  could  have  remained  in  a  moist 
condition  on  the  mouth  parts  of  the  tick,  infectiousness  would 
have  been  retained  no  longer  than  seven  or  eight  days,  since 
under  the  conditions  surrounding  the  tick,  i.e.,  in  diffuse  light  and 
at  room  temperature,  the  virus  in  one  experiment  Hved  approxi- 
mately a  week  (from  unpublished  experiments).  It  is  quite 
certain,  however,  that  the  virus  would  not  remain  in  a  moist 
condition  on  the  mouth  parts  of  the  tick,  but,  on  the  contrary, 
that  it  would  become  dried  speedily.  Extreme  desiccation  over 
sulphuric  acid  usually  destroys  the  virus  in  from  twenty-four  to 
forty-eight  hours.  It  is  probable  that  desiccation  as  it  occurs 
under  natural  conditions  of  temperature  and  atmospheric  mois- 
ture would  not  destroy  the  virus  so  soon,  but  it  would  be  difficult  to 
believe  that  its  life  could  extend  for  fifty-four  days  under  such 
circumstances. 

The  experiments  quoted  afford,  in  addition,  some  positive 
evidence  that  the  virus  proliferates  in  the  tick.  I  have  never 
found  the  blood  of  animals  suffering  from  spotted  fever  to  be 
infective  in  doses  under  0.05  c.c,  and  usually  the  minimal  patho- 
genic dose  is  nearer  o.i  c.c.  The  tick,  nymph  i,  infected  three 
guinea-pigs  at  successive  intervals,  and  if  we  assume  that  the  blood 
by  which  the  tick  was  infected  possessed  the  highest  virulence,  and 
that  the  virus  underwent  no  proliferation  in  the  tick,  it  would  have 
been  necessary  for  the  tick  to  ingest  0.15  c.c.  of  blood,  at  least,  in 


3i6  Contributions  to  Medical  Science 

order  that  it  might  later  infect  three  guinea-pigs.  This  volume 
of  0.15  c.c.  is  greatly  in  excess  of  that  of  the  tick,  since  in  this 
instance  the  adult  was  of  unusually  small  size.  Hence  it  seems 
necessary  to  conclude  that  the  tick  becomes  infected,  that  the 
virus  proHferates  in  its  body,  and  that,  on  biting  the  guinea-pig, 
the  virus,  equivalent  in  quantity  or  virulence  to  that  contained  in 
0.05  to  0.1  c.c.  of  blood  from  an  infected  animal,  is  in  some  way 
injected. 

In  the  second  place  these  results  have  an  important  relation  to 
the  disease  as  it  occurs  in  the  Bitter  Root  Valley  and  other  localities. 
They  indicate  that  the  adult  tick  which  infects  man  in  the  spring 
does  not  of  necessity  acquire  its  own  infection  in  the  spring,  but 
may  have  acquired  it  by  biting  any  one  of  several  possible  hosts 
during  the  autumn  or  winter  when  in  the  nymphal  stage. 

It  may  with  some  reason  be  suggested  that  the  experiments 
cited  above  do  not  prove  that  these  ticks  acquired  their  infection 
when  in  the  nymphal  stage,  but,  on  the  contrary,  that  they  had 
become  infected  under  natural  conditions,  that  perhaps  they  were 
infected  as  larvae,  or  that  they  derived  their  infection  from  the 
parent  tick  through  inheritance.  The  possibihty  of  these  events 
cannot  be  denied,  although  the  fact  that  the  ticks  came  from  a 
vicinity  not  known  to  be  infected  makes  it  rather  improbable. 

Experiments  are  now  being  conducted  to  determine  the  possi- 
bilities of  infecting  the  larvae  and  of  hereditary  transmission,  and 
they  will  be  reported  at  a  future  time. 

other  experiments  with  the  "wood-tick." 
Transmission  of  Rocky  Mountain  spotted  fever  by  means  of 
the  adult  female  has  already  been  reported  by  King'  and  by  me 
(p.    278),*   and  I  have  also  reported   (p.   299)^    transmission  by 
means  of  the  adult  male  tick. 

By  means  of  ticks  obtained  from  Montana  in  the  early  part  of 
1907  I  have  repeated  these  experiments  many  times.  At  present 
I  shall  only  relate  the  main  facts  concerning  the  positive  results, 
adding  the  statement  that  many  irregularities  have  been  met  with 

'  Public  Ueallh  Reports,  July  26,  1906. 
'Jour.  Am.  Med.  Assn.,  1906,  47,  p.  358. 
>  Jour.  Inject.  Dis.,  i,  1907,  4,  p.  141. 


Role  of  the  Wood-Tick  in  Spotted  Fever  317 

and  that  the  ease  with  which  individual  adults  become  infected 
is  subject  to  considerable  variation. 

Male  tick  7  produced  a  course  of  fever  in  guinea-pig  339  which  lasted  for  seven 
days.  The  animal  recovered  and  one  month  later  died  of  a  bacterial  infection.  Fe- 
male tick  4  fed  on  an  infected  monkey  (22)  for  six  hours  and  forty  minutes,  January 
12,  1907.  Subsequent  tests  of  a  thorough  character  on  guinea-pig  315  during  the  next 
month  demonstrated  that  the  tick  had  not  become  infected  from  the  monkey.  Feb- 
ruary 9  the  tick  fed  on  an  infected  guinea-pig  (333)  for  twenty-two  hours.  Nine 
days  later  it  was  allowed  to  feed  on  a  normal  guinea-pig  (381)  for  twenty-two  hours. 
This  animal  died  in  two  weeks  with  anatomic  changes  which  were  not  characteristic 
of  spotted  fever  and  the  test  was  thrown  out.  A  second  test  was  made  February  26 
(guinea-pig  404),  the  animal  showing  a  course  of  fever,  and  at  its  death  in  nine  days, 
anatomic  changes  which  are  characteristic  of  spotted  fever  in  the  guinea-pig.  Other 
successful  tests  were  made  on  guinea-pig  417,  March  7,  and  later  in  conjunction  with 
other  ticks.  Guinea-pig  415  was  bitten  by  female  ticks  4  and  7.  After  an  incubation 
period  of  forty-eight  hours  fever  developed,  and  the  animal  died  in  five  days  after  the 
ticks  were  first  attached.  A  similar  result  has  been  obtained  several  times  by  per- 
mitting more  than  one  infected  tick  to  bite  a  guinea-pig,  hence  it  is  very  probable  that 
the  virulence  of  an  infection  has  a  relation  to  the  number  of  infected  ticks  which 
feed  on  the  animal. 

Female  tick  7  did  not  become  infected  after  feeding  on  monkey  22  for  fourteen 
hours,  as  shown  by  a  test  on  guinea-pig  320,  and  on  other  pigs  during  the  course  of 
the  next  month.  February  10  it  was  allowed  to  feed  on  an  infected  guinea-pig,  the 
duration  of  the  feeding  not  being  noted.  Ten  days  later  the  disease  was  transmitted 
to  guinea-pig  386,  on  which  the  tick  fed  for  thirty  hours. 

Female  tick  8  did  not  become  infected  after  feeding  on  monkey  22  for  eight  and 
three-fourths  hours.  Two  subsequent  attempts  to  infect  this  tick  failed,  one  feeding 
having  lasted  twenty-four  hours  and  the  other  fifteen  hours.  Similar  experiences 
have  been  encountered  with  several  females  and  with  many  males.  Female  8  subse- 
quently died  in  a  blackened  state,  when  filled  with  blood. 

Females  12,  14,  15,  17,  18,  19,  and  20  were  infected  by  single  feedings  which 
lasted  from  twenty  to  thirty  hours.  This  was,  of  course,  proved  by  tests  on  normal 
guinea-pigs. 

The  experience  of  the  past  winter  has  shown  that  it  is  much 
more  difficult  to  infect  the  adult  male  than  the  female  tick.  This 
may  have  a  direct  relation  to  the  fact  that  the  male  is  less  voracious 
in  its  feeding  than  the  female,  that  it  ingests  a  smaller  quantity  of 
blood,  and  hence  has  less  chance  of  becoming  infected. 

Concerning  the  mechanism  by  which  the  tick  produces  infection, 
it  is  possible  to  produce  only  hypotheses  at  this  time.  That  the 
tick  injects  a  toxic  secretion  into  the  skin  is  manifest  from  the 
erythema  and  softening  of  the  epidermis  which  accompany  the 
bite.     Not  infrequently  a  small  area  of  tissue  sloughs  after  the  tick 


31 8  Contributions  to  Medical  Science 

is  removed.  In  all  probability  the  secretion  of  the  salivary  glands 
is  responsible  for  these  local  manifestations.  It  is  certain  that  the 
latter  have  no  relation  to  spotted  fever,  since  they  are  produced 
by  normal  as  well  as  by  infected  ticks.  In  all  the  experiments 
so  far,  both  by  the  artificial  inoculation  of  blood  and  by  the  bite  of 
the  tick,  there  is  no  reason  to  believe  that  the  disposition  of  the 
virus  produces  a  visible  local  reaction  in  the  subcutaneous  tissue. 
That  the  virus  of  spotted  fever  is  introduced  by  the  tick  with 
the  salivary  secretion  is  the  most  plausible  assumption,  although 
the  possibility  of  infection  by  regurgitation  from  the  alimentary 
tract  remains  for  consideration. 

ADDITIONAL  NOTES   ON   THE   LIFE-HISTORY   OF   THE   WOOD-TICK. 

Any  observations  throwing  light  on  the  life-cycle  of  the  wood- 
tick  (Dermacentor  occidentalis)  are  important,  because  of  the 
relation  which  this  arachnid  bears  to  the  spotted  fever. 

In  a  previous  article  (p.  291)^  I  gave  a  short  account  of  the  life- 
history  of  the  wood-tick  as  it  was  observed  under  artificial  conditions 
in  a  single  instance.  The  life-cycle  was  observed  from  the  time  the 
female  began  to  lay  her  eggs,  June  21,  until  the  young  had  emerged 
from  their  second  moult  as  young  adult  ticks  about  September  10, 
a  period  of  somewhat  less  than  three  months.  Through  accidents 
it  was  not  possible  to  preserve  the  new  brood  until  they  had  ripened 
sexually,  hence  to  this  extent  there  exists  a  temporary  gap  in  my 
observations.  Inasmuch  as  adult  ticks  are  rarely  found  in  the 
autumn  in  the  Bitter  Root  Valley,  it  seems  evident  that  the  com- 
paratively rapid  evolution  of  the  cycle  in  the  experiment  cited  was 
due  to  the  presence  of  a  host  constantly  and  to  the  rather  warm 
temperature  under  which  the  experiment  was  conducted. 

In  order  to  determine  with  some  exactness  the  form  in  which  the 
tick  survives  the  winter  and  under  what  conditions,  the  Bitter 
Root  Valley  was  visited  in  December,  1906.  Various  theories 
exist,  locally,  in  reference  to  this  point,  the  two  more  prevalent 
being,  first,  that  the  arachnid  secretes  itself  under  stones,  beneath 
the  bark  of  dead  trees,  or  that  it  burrows  into  rotten  stumps  to 

'  "Further  Observations  on  Rocky  Mountain  Spotted  Fever  and  Dermacentor  occidentalis,"  Jour. 
Am.  Med.  Assn.,  1906,  47,  p.  1067. 


Role  of  the  Wood-Tick  in  Spotted  Fever  319 

remain  there  until  spring;  second,  that  ticks  may  be  found  on 
horses  and  cattle  in  the  hills  during  almost  any  month  of  the 
winter. 

Both  possibihties  were  investigated,  the  former  resulting 
largely  in  failure.  One  individual  who  felt  perfectly  confident 
concerning  the  first  theory  failed  to  discover  ticks  in  the  suspected 
locahties  after  several  days'  search,  and  my  own  attempts  were  as 
fruitless.  That  the  tick  may  occasionally  be  found  apart  from 
living  animals  in  midwinter  was  shown  by  a  miner  who,  the  day 
before  I  saw  him,  had  found  one  in  the  tunnel  of  his  mine. 

The  second  theory  soon  found  ample  verification,  and  through  the 
kindness  of  Mr.  F.  D.  Nichols,  of  Hamilton,  and  Dr.  E.  F.  Dodds 
and  Professor  M.  J.  Elrod,  of  Missoula,  I  received  during  January 
and  February  several  hundred  ticks  which  had  been  picked  from 
horses  brought  in  from  the  hills  for  this  purpose.  Among  those 
received  early  in  January  nearly  all  were  in  the  second  moulting 
stage  and  after  varying  periods  emerged  as  adult  ticks.  With 
them,  however,  were  four  nymphs  which  had  fed  moderately  and 
were  more  or  less  distended,  and  a  few  which  had  cast  off  the  second 
skin.  The  nymphs  were  those  used  in  the  experiments  cited 
above.  During  February  all  the  ticks  received  were  either  in  the 
second  moulting  stage  or  had  emerged  from  it. 

Early  in  March  I  obtained  ffom  Mr.  Nichols  about  thirty 
adult  females  which  were  greatly  enlarged,  and  some  of  which 
began  the  deposition  of  eggs  within  a  few  days. 

In  the  laboratory  experiment  cited  above  it  was  noted  that  the 
ticks  left  the  guinea-pig  when  they  had  developed  sufficiently  to 
moult.  This  appears  not  to  be  the  rule,  however,  since  a  very 
large  percentage  of  the  ticks  which  were  in  the  second  moult  when 
received  still  grasped  lamellae  of  dried  epidermis  with  their  man- 
dibles, the  softened  epidermis  having  been  torn  loose  at  the  time 
the  ticks  were  removed. 

It  seems  probable  that  the  appearance  of  the  "wood-tick"  of 
Montana  in  an  overwhelming  "crop"  during  March,  April,  and  May, 
and  its  apparent  absence  during  other  periods  are  the  results  of 
chmatic  conditions,  and  that  it  is  prevented  from  being  a  perennial 
pest  only  on  account  of  these  conditions.     It  seems  to  be  only 


320  Contributions  to  Medical  Science 

ignorance  of  the  nature  of  the  larval,  nymphal,  and  moulting 
forms  which  has  created  the  impression  in  the  Bitter  Root  Valley 
that  the  tick  disappears  entirely  in  July  to  reappear  only  when 
spring  comes.  Description  of  these  forms  to  residents  has  frequently 
shown  that  they  have  been  observed,  although  not  identified. 

Examples  of  exceptional  development  have  been  reported  to 
me  a  number  of  times,  such  as  the  precocious  appearance  of  a  few 
adult  ticks  in  the  late  summer  or  fall.  I  was  shown  a  larval  tick 
which  had  attached  itself  to  the  ear  of  a  child  in  December,  and  in 
April  a  nymph  obtained  from  a  similar  source.  It  may  be  assumed 
that  the  larva  seen  in  December  represented  eggs  which  were 
laid  late  in  the  summer  or  in  the  fall  rather  than  in  the  early  sum- 
mer, and  that  the  nymph  seen  in  April  came  from  an  egg  which  had 
been  deposited  unusually  early  in  the  spring  or  unusually  late  in 
the  fall.  These  examples  of  precocious  or  tardy  appearance  of 
the  tick  may  well  have  a  relation  to  the  rare  occurrence  of  spotted 
fever  so  late  as  August,  or  so  early  as  February,  rather  than  during 
the  months  of  spring  at  which  time  the  disease  prevails  habitually. 

It  is  difficult  for  the  residents  of  the  Bitter  Root  Valley  to 
believe  that  the  tick  lives  exclusively  as  a  parasite  on  animals, 
on  account  of  the  widespread  and  more  or  less  uniform  distribution 
of  the  arachnid  in  nature.  It  is  thought  it  must  live  to  some 
extent  at  least  on  vegetable  or  on  dead  insect  material.  There  is 
as  yet  no  direct  evidence  to  support  this  suspicion. 

It  is  true  that  among  the  foothills  the  tick  seems  to  be  omni- 
present, yet  the  uniformity  of  its  distribution  even  in  these  locali- 
ties is  open  to  doubt.  It  is  not  uncommon  to  learn  from  a  ranch- 
man that  more  ticks  may  be  found  on  one  hill  than  on  another 
which  is  in  close  proximity.  Horses  and  cattle  roam  practically  all 
the  foothills  to  some  extent,  especially  during  the  fall  and  winter. 
During  their  residences  in  such  places  they  collect  innumerable 
ticks,  probably  in  the  form  of  larvae  chiefly.  The  young  ticks 
grow  and  moult  on  the  host  and  when  they  reach  adult  form  one 
would  be  safe  in  assuming  that  large  numbers  drop  off,  or  are 
rubbed  of!  against  bushes  and  small  pines  by  the  animals.  A  high 
wind  may  be  an  important  factor  in  disseminating  ticks,  for  a 
ranchman  related  his  experience  in  driving  home  one  evening  in 


Role  of  the  Wood-Tick  in  Spotted  Fever  321 

the  spring  during  a  violent  windstorm.  He  felt  particles  striking 
his  face  and  clothing,  and  on  reaching  home  found  a  large  number 
of  ticks  on  his  clothing.  From  my  own  experience  I  can  state  that 
ticks  climb  small  pines  to  at  least  the  full  height  of  a  man.  It  is 
probable  that  they  ascend  higher  than  this,  and  they  are  so  light 
that  without  doubt  they  could  be  easily  carried  by  a  strong  wind 
for  some  distance. 

It  is  another  fact  of  importance  that  the  tick  has  increased 
greatly  in  numbers  since  the  valley  has  been  well  settled  and  the 
quantity  of  the  stock  has  increased.  This  is  agreed  to  by  all  who 
have  lived  long  in  the  valley. 

CONDITIONS   IN   THE   LO   LO   VALLEY. 

The  conditions  in  the  Lo  Lo  Valley  during  the  past  two  years 
are  very  important.  Lo  Lo  Creek  is  tributary  to  the  Bitter  Root 
River,  and  its  valley  for  some  years  has  been  considered  as  badly 
infected.  In  1906  this  locality  had  no  infections,  and  there  was 
hope  that  the  disease  had  died  out  there.  If  the  disease  is  kept 
alive  by  ticks  which  have  bitten  infected  men,  there  would  be 
some  ground  to  hope  that  a  year  without  an  example  of  infection 
in  man  might  result  in  the  disappearance  of  the  disease.  If  after 
a  year  of  no  infections  cases  did  appear  in  the  following  year, 
three  possibilities  present  themselves  in  the  way  of  explanations: 
(i)  Certain  ticks  may  live  for  two  years  instead  of  one  year,  the 
latter  being  the  usual  period  of  life;  (2)  the  virus  may  be  transmit- 
ted through  two  generations  of  the  tick  without  its  renewal  from  an 
infected  host;  (3)  each  generation  of  ticks  may  acquire  the  disease 
afresh  each  year  from  some  susceptible  host  other  than  man  (see 
below).  There  is  not  sufficient  ground  at  present  for  considering 
a  fourth  possibility,  that  the  tick  becomes  infected  from  decaying 
organic  matter. 

As  stated,  no  cases  occurred  in  the  Lo  Lo  Valley  during  1906, 
and  but  one  has  appeared  in  1907.  It  is  hoped  that  the  first 
two  possibilities  can  be  decided  experimentally. 

SUSCEPTIBILITY   OF   THE    GOPHER   AND   OTHER   ANIMALS. 

The  third  possibility  mentioned  above,  that  spotted  fever  may 
be  kept  alive  in  a  susceptible  host  other  than  man,  has  received 


322  Contributions  to  Medical  Science 

some  support  this  year.  Assisted  by  Mr.  P.  G.  Heinemann,  I 
have  found  that  the  indigenous  gopher  or  ground  squirreP  may  be 
infected  by  inoculation  and  that  one  attack  confers  immunity  to 
second  inoculations.  Its  susceptibility  is  much  less  than  that 
of  the  guinea-pig  and  is  not  perfectly  uniform.  The  disease  was 
again  conveyed  to  the  guinea-pig  from  the  inoculated  gophers. 
I  do  not  maintain  at  this  time  that  the  gopher  exclusively  is  respon- 
sible for  the  maintenance  of  the  disease,  although  this  is  possible. 
The  susceptibility  of  other  animals  found  in  the  valley  is  being 
investigated.  Dr.  William  M.  Chowning  co-operated  with  us 
in  the  inoculation  of  a  horse,  which  received  about  60  c.c.  of  blood 
subcutaneously  from  infected  guinea-pigs.  After  a  brief  incuba- 
tion period,  the  animal  ran  a  short  but  rather  high  course  of  fever. 
The  horse  resisted  entirely  a  second  inoculation  of  60  c.c.  of  infected 
blood  from  man  and  monkey,  the  experiment  being  conducted 
by  Mr.  Heinemann  and  myself.  Assisted  by  Mr.  Heinemann, 
a  second  horse  was  inoculated  with  10  c.c.  of  infected  blood  from 
man.  No  distinct  reaction  followed.  A  calf,  2  weeks  old,  failed 
to  react  following  the  inoculation  of  over  30  c.c.  of  blood  from 
infected  guinea-pigs. 

infected  ticks  in  nature. 

Although  the  experimental  transmission  of  Rocky  Mountain 
spotted  fever  by  means  of  the  "wood-tick"  attracts  the  gravest 
suspicion  to  this  arachnid  as  the  agent  of  infection  under  natural 
conditions,  the  theory  of  tick-transmission  cannot  be  considered 
as  proved  circumstantially  until  infected  ticks  are  discovered  in 
nature.  Assisted  by  Mr.  Heinemann,  it  has  been  possible  to 
demonstrate  that  infected  ticks  do  exist  under  natural  conditions 
on  the  west  side  of  the  Bitter  Root  Valley.  The  ticks  were  col- 
lected from  vicinities  in  which  the  disease  appeared  this  year, 
partly  from  horses  and  cows  and  partly  from  the  woods. 

A  group  of  thirty-six  males  taken  from  the  vicinity  of  Mr. 
Hickey's  ranch  produced  the  disease  in  guinea-pig  596,  which 
presented  a  typical  clinical  course  with  scrotal  hemorrhages  and 
died  in   twelve   days  after   the   ticks  were  first   attached.     The 

■  The  following  experiments,  with  others,  will  be  reported  in  detail,  in  conjunction  with  Mr.  Heine- 
mann in  the  near  future. 


Role  of  the  Wood-Tick  in  Spotted  Fever  323 

anatomic  changes  were  characteristic  of  spotted  fever  in  every 
detail,  as  it  occurs  in  the  guinea-pig,  and  cultures  from  the  heart, 
liver,  and  spleen  remained  free  from  discoverable  bacterial  growth. 
I  take  the  position  thai  these  experiments  connect  the  tick 
unmistakably  with  the  occurrence  of  spotted  or  tick  fever  of  man 
in  the  Bitter  Root  Valley,  and  that  without  them  the  "tick  theory" 
would  lack  essential  support. 


FURTHER  EXPERIMENTS  WITH  THE  WOOD-TICK  IN 

RELATION  TO  ROCKY  MOUNTAIN 

SPOTTED  FEVER/ 

H.     T.     RiCKETTS. 

{From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

Experiments  which  I  previously  reported  show  that  the  adult 
male  and  female  and  the  nymph  of  the  Rocky  Mountain  wood- 
tick  (Dermacentor  occidentalis)  are  able  to  acquire  and  transmit 
spotted  fever. 

In  this  paper  experiments  will  be  reported  which  show:  (i)  that 
the  larva  may  acquire  the  disease  and  remain  infective  during  the 
nymphal  stage;  (2)  that  the  virus  may  be  transmitted  from  an 
infected  female  to  her  young  through  the  eggs;  (3)  that  the  virus 
exists  in  both  the  gut  and  the  salivary  glands  of  the  infected  tick. 

I.      INFECTION   OF   THE   LARVA. 

In  these  experiments  the  possibiHty  of  the  lar\'ae  having  acquired  the  disease 
from  the  female  parent  through  the  egg  was  excluded  by  testing  the  females  on  healthy 
guinea-pigs  before  they  deposited  their  eggs,  and  it  is  to  be  understood  that  all  the 
larvae  used  in  these  experiments  came  from  females  which  had  been  tested  in  this 
way  and  found  uninfected. 

When  from  one-third  to  three-quarters  of  the  eggs  of  one  or  more  females  had 
hatched,  the  bottle  which  contained  them  was  placed  in  a  tick-proof  cage  with  a  guinea- 
pig.  As  the  eggs  hatched,  the  living  larvae  were  removed  from  the  bottle  and  placed 
on  the  guinea-pig,  where  they  were  left  to  feed  or  to  drop  off  until  ready  to  feed. 
Within  a  few  days  the  guinea-pig  was  inoculated  with  spotted  fever  and  the  course 
of  the  fever  and  the  condition  of  the  animal  were  observed  daily.  When  the  larvae 
had  fed  sufficiently  they  fell  from  the  animal  and  crawled  up  on  the  canvas  which 
covered  the  cage.  From  this  location  thej'  were  removed  and  placed  in  boxes  to  await 
moulting  and  the  nymphal  stage,  and  when  a  sufficient  number  had  reached  this 
stage  they  were  placed  in  a  fresh  cage  with  a  healthy  guinea-pig  on  which  they  were 
allowed  to  feed. 

It  was  considered  preferable  to  carry  on  the  experiments  in  this  way,  i.e.,  to 
infect  as  larvae  and  to  test  as  nymphs,  rather  than  to  attempt  both  steps  during  the 
larval  stage.  The  latter  course  would  have  involved  serious  difficulties,  and,  more- 
over, the  results  have  a  more  practical  bearing  when  it  is  shown  that  the  tick,  having 
acquired  the  disease  as  a  larva,  remains  infective  after  reaching  the  nymphal  stage. 

I  From  Jour.  Am.  Med.  Assn.,  1907,  49,  p.  1278.  This  work  has  been  supported  by  an  appropria- 
tion made  by  the  legislative  assembly  of  the  state  of  Montana,  at  the  solicitation  of  the  Montana 
State  Board  of  Health,  and  aid  has  also  been  rendered  by  the  University  of  Chicago,  and  by  the  Memorial 
Institute  for  Infectious  Diseases,  Chicago. 

324 


Experiments  with  Wood-Tick  in  Spotted  Fever      325 

Larval  infection  2. — On  July  8  several  thousand  normal  larvae 
were  placed  with  an  infected  guinea-pig  (670).  A  large  number 
fed,  dropped  off,  and  were  isolated,  and  when  they  began  to  moult 
were  placed  with  a  normal  guinea-pig  (717).  After  a  period  of 
six  days  the  temperature  of  the  latter  rose  to  105.7  F.,  and  on 
subsequent  days  it  registered  105,  106,  105.4,  and  105  F.  The 
animal  was  found  dead  on  July  20,  after  six  days  of  fever  and  twelve 
days  after  it  was  placed  with  the  ticks. 

The  condition  at  autopsy  was  typical  for  spotted  fever  in  the 
guinea-pig.  The  spleen  was  enormously  enlarged,  fairly  firm, 
cyanotic,  and  granular.  The  inguinal,  axillary,  and  mesenteric 
lymph  glands  were  deeply  congested  or  hemorrhagic,  and  the 
surrounding  areolar  tissue  was  congested.  The  kidneys  and 
suprarenal  glands  were  enlarged  and  congested.  The  liver  was 
somewhat  enlarged.  The  heart  and  lungs  appeared  normal.  No 
hemorrhages  had  occurred  in  the  external  genital  organs.  The 
guinea-pig  was  a  female,  and  hemorrhages  are  less  constant  in  the 
vulva  than  in  the  scrotum.  Cultures  on  agar  slants  made  from 
the  heart,  spleen,  and  kidney  remained  free  from  growth.  Twelve 
enlarged  nymphs  were  removed  from  the  pig  and  cage,  and  this 
may  be  taken  as  a  fair  index  of  the  number  which  had  fed. 

Guinea-pig  732  likewise  was  infected  by  the  remaining  nymphs, 
the  animal  making  an  eventual  recovery. 

Of  the  several  thousand  larvae  placed  with  the  infected  guinea- 
pig  (670)  only  a  small  proportion  fed  during  the  course  of  the  fever. 
Under  the  title  of  larval  infection  7  the  remaining  larvae  were 
exposed  to  infection  on  guinea-pig  637,  which  was  inoculated  on 
the  fourth  day  after  being  placed  with  the  larvae.  This  animal 
had  been  used  previously  in  testing  ticks  found  in  nature,  but  had 
not  been  infected,  a  fact  which  was  shown  by  the  typical  course 
of  fever  which  followed  inoculation. 

The  larvae  which  fed  on  guinea-pig  637  were  collected  and 
after  they  had  reached  the  nymphal  stage  were  placed  with  a 
normal  guinea-pig  (725).  The  temperature  of  the  latter  rose  to 
104.7  F.  on  the  sixth  day  and  death  occurred  three  days  later. 
Although  this  is  a  rapid  course  for  spotted  fever,  the  anatomic 
changes  were  characteristic.      There  were  enlarged  and  intensely 


326  Contributions  to  Medical  Science 

congested  lymph  glands;  enlarged,  cyanotic,  and  rather  firm 
spleen,  and  incipient  scrotal  hemorrhages.  No  gross  changes 
were  discoverable  in  the  other  organs,  and  inoculations  of  agar 
slants  from  the  liver,  spleen,  and  heart's  blood  showed  no  growth. 
At  least  seven  nymphs  had  bitten  the  guinea-pig.  In  order  to 
corroborate  this  result  further,  a  monkey  was  inoculated  from  an 
emulsion  of  the  Hver,  spleen,  kidney,  and  suprarenal  gland.  The 
temperature  of  this  animal  on  the  second  day  rose  to  104  F.,  and 
on  subsequent  days  registered  105.8,  104.7,  105.4,  104.  i  F.,  and 
death  occurred  on  the  seventh  day  after  inoculation.  The  fol- 
lowing were  the  essential  changes  seen  at  autopsy:  beginning 
hemorrhages  in  the  skin  of  the  eyebrows  and  cheeks;  the  whole 
perineum  was  cyanotic  and  showed  a  roseolar  eruption;  the 
lymph  glands  everywhere  were  enlarged  and  congested  but  were 
not  hemorrhagic;  the  spleen  was  greatly  enlarged,  rather  firm  and 
cyanotic.  No  changes  could  be  noted  in  other  organs.  The 
virus  was  again  passed  through  another  guinea-pig  which  reacted 
typically  and  the  strain  was  then  dropped. 

Still  another  test  was  made  with  the  nymphs  which  remained. 
After  an  incubation  period  of  five  days  the  guinea-pig  (739)  showed 
a  temperature  of  105.3  F.  and  died  six  days  later  with  char- 
acteristic anatomic  changes.  No  ordinary  microbes  could  be 
cultivated  from  the  tissues.  At  least  six  nymphs  had  fed  on  the 
guinea-pig. 

Larval  infection  4. — On  July  10  about  2,000  normal  larvae 
were  placed  with  guinea-pig  659  and  a  few  days  later  the  animal 
was  infected  by  inoculation.  The  guinea-pig  died  on  July  21  and 
was  replaced  by  another  (688)  which  was  infected  similarly  and 
died  on  August  2.  A  third  guinea-pig  (714)  was  introduced  on 
this  date,  was  inoculated  on  August  5,  and  died  August  14.  All 
three  animals  presented  the  course  and  anatomic  changes  of 
spotted  fever. 

The  larvae  which  had  fed  were  collected  and,  after  hatching 
as  nymphs,  360  were  placed  with  a  normal  guinea-pig  (726),  the 
nymphs  being  added  as  they  moulted.  None  of  the  nymphs 
became  attached  for  seven  to  ten  days,  and  about  ten  days  after 
feeding  was  discovered  a  course  of  fever  began  in  the  guinea-pig 


Experiments  with  Wood-Tick  in  Spotted  Fever      327 

and  the  latter  died  in  six  days,  showing  the  characteristic  anatomic 
changes  of  spotted  fever. 

In  a  similar  manner  the  nymphs  of  larval  infection  6  killed 
two  guinea-pigs  successively  (731  and  751),  and  the  combined 
n>-mphs  of  experiments  2  and  7  produced  spotted  fever  in  guinea- 
pig  674,  which  eventually  recovered. 

II.     transmission  of  the  virus  from  the  infected  female 
to  her  young,  through  the  egg. 

On  hj-pothetical  grounds  these  so-called  heredity  experiments  were  performed 
in  two  groups:  First,  certain  females  were  exposed  to  infection  before  they  were 
impregnated,  by  permitting  them  to  feed  on  guinea-pigs  which  had  been  inoculated 
with  spotted  fever.  This  was  done  in  experiments  3  and  5.  Second,  the  ticks  of 
experiments  4  and  6  had  been  impregnated  before  their  infection  was  attempted. 
This  course  was  pursued,  since  it  has  been  suggested  in  the  literature  that  failure 
in  hereditary  transmission  may  depend  on  the  time  of  infection  with  relation  to  the 
impregnation  of  the  female.  After  impregnation  many  of  the  ova  begin  to  enlarge, 
and,  if  the  female  is  provided  with  food,  they  become  surrounded  by  a  denser  pellicle. 
Presumably  a  microbe  could  penetrate  such  eggs  less  readily  than  the  more  minute 
and  more  delicate,  immatiure  ova  or  germ  cells  of  the  unimpregnated  female. 

In  this  instance,  however,  the  point  just  discussed  is  not  an  essential  one,  since 
the  most  satisfactory  results  which  I  obtained  involved  females  which  were  impregnated 
before  their  infection  was  attempted.  Casual  observation  of  the  manner  in  which 
the  eggs  mature  makes  it  clear  why  the  conception  mentioned  is  to  a  certain  extent 
irrelevant  in  so  far  as  the  probability  of  infection  of  the  eggs  is  concerned.  If  one 
exposes  the  viscera  of  a  fully  developed  female  which  is  on  the  point  of  laying  its 
eggs  it  is  seen  that  only  a  small  proportion  of  the  ova  have  matured,  i.e.,  have  reached 
the  limit  of  size  and  the  appearance  which  they  habitually  obtain  before  extrusion. 
Hundreds  of  smaller  and  still  smaller  ova  are  seen  in  the  sac  as  one  follows  it  back,  a 
fact  which  makes  it  apparent  that  the  eggs  are  matured  seriatim,  the  food  which  is 
found  in  the  greatly  distended  aUmentary  sac  being  gradually  consumed  in  the  mean- 
time. Hence,  even  in  the  thoroughly  ripened  female  one  finds  many  minute  and 
delicate  ova  which  apparently  would  be  susceptible  to  penetration. 

None  of  the  females  used  in  these  experiments  was  infected  in  the  first  instance. 
They  were  among  ticks  which  had  been  collected  from  animals  and  from  the  woods 
during  the  search  for  infected  ticks  in  nature,  and  each  had  fed  on  a  normal  guinea- 
pig  for  a  period  much  longer  than  that  required  for  the  transmission  of  the  disease. 

If  the  female  failed  to  enlarge  after  prolonged  feeding  and  after  the  passage  of 
abundant  feces  the  result  was  considered  as  proof  that  she  had  not  been  impregnated. 

It  is  not  my  intention  to  analyze  all  the  experiments  in  detail  at  this  time.  In 
addition  to  reporting  two  positive  transmissions  through  the  egg,  I  may  mention  the 
fact  that  short  courses  of  fever  were  produced  by  the  larvae  in  several  instances, 
without  the  development  of  cutaneous  phenomena.  Immunity  tests  are  still  to  be 
made  with  such  animals,  in  order  to  determine  whether  these  occurrences  represented 
mild  attacks  of  spotted  fever. 

The  technic  was  similar  to  that  used  in  the  experiments  termed  larval  infections, 


328  Contributions  to  Medical  Science 

i.e.,  the  lan-ae,  as  soon  as  hatched,  were  placed  with  normal  guinea-pigs  in  tick-proof 
cages. 

Both  experiments  concern  heredity  experiment  4,  in  which  infection  of  the  ticks 
took  place  subsequent  to  their  impregnation;  that  is  to  say,  the  ticks  were  not  allowed 
to  feed  on  infected  guinea-pigs  until  the  character  of  their  enlargement  after  feeding 
on  normal  guinea-pigs  indicated  that  they  had  been  impregnated.  The  rather  scant 
supply  of  guinea-pigs  rendered  it  impossible  to  prove  the  infectivity  of  all  females 
before  the  eggs  were  laid.  This  was  done  in  several  instances,  however,  and  all  which 
were  so  tested  proved  to  be  infected.  Concerning  the  two  e.xperiments  to  be  reported, 
the  infectivity  of  the  larvae  is  ample  proof  that  the  females  had  been  previously 
infected. 

The  precaution  was  taken  to  perform  the  experiments  in  entirely  new  cages,  so 
that  the  larvae  had  no  opportunity  to  come  in  contact  with  spotted  fever  virus  other 
than  that  contained  in  the  female  parent. 

Females  7  and  9  of  heredity  experiment  4  fed  on  four  infected  guinea-pigs  from 
May  22  until  June  14,  and  at  this  time  they  were  ready  for  o\nposition. 

Experiment  with  female  7. — This  female  was  removed  from  an 
infected  guinea-pig  June  14;  it  began  laying  eggs  June  27;  hatching 
began  on  or  about  July  27.  It  was  estimated  that  about  2,000 
eggs  were  laid. 

Beginning  on  July  30,  the  larvae,  as  they  hatched,  were  placed 
with  a  normal  guinea-pig  (707).  Inasmuch  as  they  do  not  feed 
for  some  days  after  hatching,  immediate  results  could  not  be 
anticipated. 

On  August  22  or  23  the  temperature  of  the  guinea-pig  rose  to 
104.5  F.,  the  following  day  to  105.5  F.,  in  the  vicinity  of  which 
point  it  remained  until  the  animal  died  August  28.  The  following 
notes  were  made  at  autopsy:  The  lymph  glands  are  deeply 
congested  and  considerably  enlarged  and  the  surrounding  areolar 
tissue  is  congested.  The  spleen  is  about  twice  its  normal  size, 
is  fairly  cyanotic  and  moderately  firm,  resembling  the  spleen  of 
spotted  fever  when  death  occurs  at  an  early  stage.  The  kidneys 
are  congested  and  degenerated,  and  the  suprarenals  are  congested 
and  enlarged.  The  appendages  of  the  testicles  show  a  good  deal 
of  congestion.  There  is  no  enlargement  of  the  scrotum  and  no 
hemorrhages  have  occurred  into  it.  (I  have  stated  hitherto  that 
these  scrotal  changes  are  sometimes  missed  when  death  occurs 
early.)     Other  organs  appear  to  be  unchanged. 

Agar  slants  inoculated  from  the  heart's  blood,  from  the  liver 
and  the  spleen,  remained  free  from  discoverable  growth. 


Experiments  with  Wood-Tick  in  Spotted  Fever      329 

Two  hundred  and  eleven  enlarged  larvae  were  removed  from 
the  cage,  hence  this  number  at  least  had  bitten  the  guinea-pig. 

In  order  to  verify  the  suspicion  that  this  animal  had  died  of 
spotted  fever,  an  emulsion  of  its  spleen  and  liver  was  injected 
into  guinea-pig  746.  The  temperature  of  this  animal  was  recorded 
as  follows  on  successive  days:  103.7,  102.7,  104I)  io5-5j  105 -6, 
105.4,  105.8,  105.3,  104.8,  104,  and  102.2  F.  Death  occurred 
on  the  fourteenth  day  following  inoculation. 

AUTOPSY  notes. 

Lymph  glands  are  enlarged  and  deeply  congested  or  hemor- 
rhagic; spleen  is  greatly  enlarged,  cyanotic,  and  fairly  firm;  kidneys 
are  swollen  and  congested;  the  scrotum  is  greatly  enlarged  and 
intensely  hemorrhagic,  and  the  tunica  vaginalis  is  deeply  congested. 
No  gross  changes  are  seen  in  other  organs. 

The  diagnosis  of  spotted  fever  was  made  positively. 

Experiment  with  female  g. — This  female  was  removed  from  an 
infected  guinea-pig  June  14;  it  began  laying  eggs  June  28;  hatch- 
ing began  about  July  28.     Several  hundred  eggs  were  laid. 

Beginning  on  July  31  the  larvae  were  placed  with  a  normal 
guinea-pig  (710)  as  rapidly  as  they  hatched.  Ticks  were  first 
seen  attached  after  thirteen  days  and  the  first  enlarged  larvae 
were  removed  on  the  fifteenth  day.  Ten  or  eleven  days  later  the 
temperature  of  this  animal  rose  and  continued  within  the  limits 
of  104.3  ^^J^d  105 -3  F-  fo^  seven  or  eight  days.  Following  this 
the  fever  subsided  and  the  animal  recovered.  Slight  enlargement 
of  the  scrotum  after  several  days  of  fever  was  the  only  condition, 
aside  from  the  temperature,  which  suggested  spotted  fever. 

Inasmuch  as  in  some  other  animals  in  these  experiments  the 
disease  had  run  a  similar  course,  with  eventual  recovery,  it  was 
suspected  that  the  condition  represented  a  mild  attack  of  spotted 
fever.  In  order  to  decide  this  point  if  possible,  3  c.c.  of  blood  were 
drawn  from  the  heart  of  guinea-pig  710  during  the  height  of  fever 
and  injected  into  a  normal  guinea-pig  (749).  The  following 
course  of  the  temperature  in  this  animal  was  observed  on  succes- 
sive days:  102.4,  103.4,  103.4,  105.6,  104.8,  106.7,  106.4,  106, 
104.6,   105.4,   104.4,   I04;  104.2,  and  103.5  F.     The  guinea-pig 


330  Contributions  to  Medical  Science 

recovered.  On  the  fourth  day  the  scrotum  began  to  swell,  and  on 
the  tenth  day  the  swelling  was  extreme  and  extensive  hemorrhages 
had  appeared.  With  recovery  the  hemorrhagic  areas  in  the 
scrotum  became  gangrenous,  and  following  separation  of  the 
sloughs  a  deformed  and  cicatricial  scrotum  remained. 

A  positive  diagnosis  of  spotted  fever  in  this  animal  was  made, 
and  the  diagnosis  was  confirmed  later  by  a  severe  immunity  test, 
in  which  no  reaction  occurred. 

Approximately  two  hundred  larvae  had  bitten  guinea-pig  710. 

The  results  of  these  two  experiments  prove,  without  question, 
that  the  virus  of  spotted  fever  may  pass  from  an  infected  female 
to  her  young  through  the  egg.  The  practical  importance  of  this 
fact  will,  to  a  certain  extent,  depend  on  the  viability  of  the  virus 
in  larvae  which  have  been  infected  in  this  manner;  this  is  to  say: 
Will  such  ticks  be  infective  in  their  subsequent  n^nnphal  and 
adult  stages?  Although  one  probably  should  not  try  to  forecast 
results,  the  positive  results  which  I  have  obtained  in  infecting 
normal  larvae  and  nymphs  suggest  that  larvae  which  have  acquired 
their  disease  through  the  egg  will  also  prove  infective  when  they 
have  reached  the  adult  stage.  This  suspicion  is  particularly  well 
founded  since  the  process  of  moulting  does  not  destroy  the  infec- 
tion in  either  the  larva  or  the  nymph. 

II.    the  infectivity  of  the  salivary  glands  and  the  alimen- 
tary  SAC   OF   THE  diseased   TICK, 

I  wish  to  report  a  single  experiment  in  which  the  salivary  glands 
and  the  alimentary  sac  of  an  infected  male  tick  (15)  were 
dissected  out  and  injected  separately  into  normal  guinea-pigs. 
Male  tick  15  had  been  infected  by  feeding  on  a  diseased  guinea-pig 
and  its  infectivity  was  proved  on  normal  guinea-pig  677. 

One  cannot  well  remove  the  alimentary  sac  without  contaminat- 
ing all  other  viscera  with  its  contents.  On  the  other  hand,  one 
can  grasp  a  salivary  gland  with  forceps  near  the  excretory  orifice 
and,  after  a  little  careful  dissection,  remove  the  entire  gland  with- 
out rupturing  it. 

Hence  in  this  experiment  the  salivary  glands  were  removed 
first  and  without  rupturing  the  alimentary  sac,  after  which  the 


Experiments  with  Wood-Tick  in  Spotted  Fever      331 

latter  was  removed  without  regard  to  remaining  structures.  The 
glands  and  the  sac  were  then  triturated  separately  in  sterile  salt 
solution  and  injected,  the  salivary  glands  being  inoculated  into 
guinea-pig  683  and  the  alimentary  sac  into  694. 

From  the  date  of  inoculation,  July  15,  the  following  record  of 
the  temperature  of  guinea-pig  683  was  made  on  successive  days: 
104,  102.4,  102.3,  io5-7>  105.7,  105.8,  105.9,  105. I,  105.4, 
105.4,  104. 1,  103.2,  102. 1,  102.6,  102.7  F.,  etc.  There  was 
eventual  recovery.  Four  days  after  inoculation  the  scrotum 
began  to  swell  and  on  the  seventh  day  it  was  extremely  hemor- 
rhagic and  enlarged.  On  recovery  the  hemorrhagic  areas  sloughed. 
The  course  of  fever  and  scrotal  changes  in  guinea-pig  684  were 
similar,  though  not  so  extreme. 

On  August  8  both  animals  were  given  immunity  tests  with 
three  cubic  centimeters  of  infected  blood,  a  quantity  which  con- 
tained from  thirty  to  sixty  minimum  pathogenic  doses.  Neither 
animal  showed  any  reaction  to  the  second  inoculation. 

In  a  repetition  of  this  experiment  two  ticks  should  be  used, 
the  salivary  glands  being  taken  from  one  and  the  alimentary  sac 
from  the  other. 

It  seems  probable  that  the  virus  passes  from  the  alimentary 
sac  of  the  tick  to  the  salivary  glands;  possibly  this  can  be  deter- 
mined experimentally.  Indeed,  since  the  oviducts  and  the  eggs 
of  the  female  are  invaded,  at  least  in  certain  instances,  it  is  not 
unlikely  that  spotted  fever  exists  as  a  generalized  infection  in  the 
tick  for  a  greater  or  less  period.  Whether  it  eventually  becomes 
localized  in  certain  organs  is  a  question  for  future  investigation. 
However  this  may  be,  the  disease  is  often,  if  not  always,  a  com- 
paratively harmless  incident  for  the  tick. 

SUMMARY. 

I  have  established  the  following  points  concerning  the  relation- 
ship of  the  Rocky  Mountain  wood-tick  to  the  spotted  fever  of 
western  Montana: 

1 .  Infected  ticks  exist  in  the  so-called  infected  districts  in  nature. 

2.  Both  the  adult  male  and  the  adult  female  may  acquire  the 
disease  by  feeding  on  an  infected  animal,  and  may  transmit  it 


332  Contributions  to  Medical  Science 

to  a  normal  susceptible  animal  for  a  period  of  several  weeks  there- 
after. 

3.  During  either  of  its  intermediate  active  stages,  larval  or 
nymphal,  the  tick  may  acquire  the  disease  in  the  same  manner, 
retain  it  during  moulting,  and  prove  infective  when  it  reaches 
the  subsequent  active  stage. 

4.  The  infected  female  may  transfer  the  disease  to  her  young 
through  the  egg.  It  is  possible  that  this  does  not  happen  in  all 
instances,  and  it  is  quite  certain  that  the  brood  of  an  infected 
female  may  include  many  uninfected  larvae.  This  seems  to  have 
been  proved  in  three  of  my  experiments  in  which  the  infectivity 
of  the  females  had  been  proved  before  oviposition;  the  larvae 
in  these  experiments  failed  to  infect  normal  guinea-pigs. 

5.  The  virus  exists  in  both  the  saHvary  glands  and  the  gut  of 
infected  ticks  at  a  certain  time,  and  since  it  also  invades  the  genera- 
tive organs  of  the  female  the  condition  is  probably  one  of  a  general- 
ized infection,  at  least  for  a  period.  The  disease  is  not  highly 
destructive  for  the  tick. 

At  least  two  important  steps  may  now  be  taken  in  an  aggressive 
fight  against  the  disease:  first,  a  thorough  dissemination  of  the 
knowledge  that  the  tick  is  the  agent  of  infection;  second,  a  massive 
reduction  of  the  number  of  ticks  in  infected  districts  by  means  now 
used  in  the  destruction  of  the  cattle-tick  of  the  southern  states. 
Extermination  of  the  tick  in  the  mountains  will  not  be  possible 
so  long  as  native  wild  animals  inhabit  the  soil  and  roam  the  hills. 
On  the  other  hand,  it  is  known  locally  that  the  number  of  ticks  in 
the  Bitter  Root  Valley  has  increased  enormously  as  greater  num- 
bers of  domesticated  animals  have  been  introduced,  and  the 
latter  now  seem  to  be  the  chief  hosts  for  the  tick. 


A   SUMMARY  OF  INVESTIGATIONS   OF  THE  NATURE 

AND    MEANS    OF    TRANSMISSION    OF    ROCKY 

MOUNTAIN  SPOTTED  FEVER.' 

H.     T.     RiCKETTS. 

(From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

In  the  Medical  Sentinel  (Portland,  Ore.)  for  October,  1899, 
Dr.  Edward  E.  Maxey,  of  Boise  City,  Idaho,  called  attention  to 
*'the  so-called  spotted  fever  of  Idaho."  He  described  it  as  ''an 
acute,  endemic,  non-contagious,  but  probably  infectious,  febrile 
disease,  characterized  clinically  by  a  continuous  moderately  high 
fever,  severe  arthritic  and  muscular  pains,  and  a  profuse  petechial 
or  purpuric  eruption  in  the  skin,  appearing  first  on  the  ankles, 
wrists,  and  forehead,  but  rapidly  spreading  to  all  parts  of  the 
body."  The  disease  is  not  to  be  confused  with  typhus  fever  nor 
epidemic  cerebrospinal  meningitis,  to  both  of  which  the  term 
spotted  fever  is  sometimes  applied. 

Rocky  Mountain  spotted  fever  is  not  limited  to  Idaho,  however, 
but  occurs  also  in  western  Montana,  Utah,  Nevada,  Oregon, 
Colorado,  Wyoming,  and  perhaps  in  other  neighboring  states. 
The  conditions  in  the  Bitter  Root  Valley  in  western  Montana 
aroused  such  concern  that  Dr.  Louis  B.  Wilson  and  Dr.  William 
M.  Chowning,  then  of  the  University  of  Minnesota,  undertook  an 
investigation  in  1902.^  They  learned  that  the  disease  occurs  only 
during  the  spring  and  early  summer,  and  that  the  eastern  side  of 
the  Bitter  Root  Valley  is  free  from  infection.  They  also  announced 
the  discovery  of  an  erythrocytic  parasite,  Piroplasma  hominis,  as 
the  cause  of  the  disease  and  promulgated  the  tick-gopher  h}po th- 
esis. According  to  this  hypothesis  the  gopher  is  the  habitual 
host  for  Piroplasma  hominis,  and  spotted  fever  is  conveyed  from 
the  gopher  to  man  by  means  of  the  bite  of  the  wood-tick  (Derma- 
centor  occidentalis) ,  which  feeds  on  both  man  and  animals.     It 

'  From  Trans.  Chic.  Path.  Soc,  1907.  This  work  was  supported  chiefly  by  an  appropriation  made 
by  the  legislative  assembly  of  Montana,  and  also  by  Missoula  and  Ravalli  counties,  Montana,  and  by  the 
University  of  Chicago. 

'  Wilson  and  Chowning,  Jour.  Infect.  Dis.,  1904,  i,  p.  31. 

333 


334  Contributions  to  Medical  Science 

was  stated  that  Piroplasma  hominis  exists  in  about  20  per  cent 
of  the  gophers  from  the  infected  side  of  the  valley  but  was  not 
found  in  those  from  the  east  side.  They  furnished  no  experimental 
evidence  in  favor  of  the  tick  theory. 

Anderson/  and  later  Stiles,^  of  the  Hygienic  Laboratory,  also 
studied  the  disease,  the  former  advocating  the  tick  theory,  whereas 
Stiles  discredited  it  and  failed  to  identify  Piroplasma  hominis. 
Neither  performed  experiments  bearing  on  the  tick  theory. 

Hence  when  I  began  my  investigations  in  the  Bitter  Root 
Valley  in  April,  1906,  the  tick  theory  was  little  more  than  a  h>^othe- 
sis,  supported  only  by  the  coincidence  of  the  season  of  the  adult 
tick  with  that  of  spotted  fever,  and  the  almost  constant  history  of 
tick  wounds  shortly  preceding  the  onset  of  the  disease.  Further- 
more, the  vaHdity  of  the  latter  point  was  considered  questionable 
in  some  quarters,  inasmuch  as  a  great  many  people  are  tick-bitten 
every  spring  without  suffering  from  spotted  fever  as  a  consequence. 
Although  this  circumstance  would  not  render  the  tick  theory 
untenable  it  has  caused  a  great  deal  of  local  prejudice  against  it. 
Locally  there  is  a  strong  disposition  to  attribute  to  the  water  of 
certain  streams  the  power  of  carrying  the  poison  to  man,  presum- 
ably from  foci  of  decaying  vegetation.  It  is  not  necessary  in  this 
place  to  go  into  detail  regarding  the  conditions  which  a  priori 
lent  to  the  water  theory  a  degree  of  plausibility. 

Since  the  study  of  spotted  fever  had  hitherto  been  limited 
to  the  season  during  which  man  is  attacked  it  appeared  of  great 
importance  to  discover  susceptible  laboratory  animals,  if  possible, 
in  order  that  experimental  work  might  proceed  without  serious 
interruption.  As  the  result  of  investigation  it  was  soon  deter- 
mined that  the  guinea-pig  and  monkey  (Macacus  rhesus)  are 
highly  susceptible,  that  rabbits  are  almost  non-susceptible,  and 
that  rats  and  mice  exhibit  no  outward  sign  of  illness  when  infection 
is  attempted.^ 

The  following  points  are  taken  as  proof  that  the  disease  repro- 
duced in  these  animals  is  spotted  fever:  (i)  The  incubation  period 
approximates  that  in  man  in  so  far  as  it  has  been  ascertained  in 

■  .\nderson,  Bull.  No.  14,  Byg.  Lab.,  U.S.  Pub.  Health  and  Mar.  Hasp.  Sen.,  Washington. 

■  Stiles,  Bull.  No.  20,  ibid. 

>  Ricketts,  Jour.  Am.  Med.  Assn.,  1906,  47,  p.  33.     (See  p.  278  of  this  volume.) 


Transmission  of  Spotted  Fever  335 

the  case  of  man;  (2)  the  course  of  fever,  the  eruption  and  anatomic 
changes  found  at  autopsy  correspond  closely  with  these  features 
in  man;  (3)  as  in  the  case  of  man  it  has  not  been  possible  to  culti- 
vate a  micro-organism  from  the  blood  or  organs  of  typical  cases; 
(4)  indefinite  transmission  from  animal  to  animal  appears  to  be 
possible  (100  generations). 

Inasmuch  as  the  disease  was  conveyed  to  the  latter  by  inoculat- 
ing them  with  the  blood  of  human  patients,  and  since  it  can  be 
transmitted  indefinitely  from  animal  to  animal  in  this  way  by  means 
of  the  blood,  two  very  important  points  regarding  the  nature  of 
the  disease  are  thereby  estabHshed:  First,  that  the  disease  is 
caused  by  a  virus  which  is  capable  of  propagation,  a  Hving  organism, 
for  otherwise  the  consecutive  inoculation  of  100  generations  of 
animals  would  not  have  been  possible;  second,  spotted  fever  is 
a  systemic  or  generalized  infection,  since  the  circulating  blood  is 
invaded  by  the  micro-organism.  Inoculation  experiments,  per- 
formed later,  showed  that  all  the  important  organs  of  diseased 
animals  are  heavily  infected.^ 

Further  experiments  relating  to  the  distribution  of  the  virus 
among  the  blood  elements  showed  the  following:^ 

1.  The  cell-free  serum  of  infected  blood  contains  large  amounts 
of  the  virus. 

2.  The  virus  is  present  in  considerable  quantities  in  an  artifi- 
cially induced  leukocytic  exudate,  free  from  erythrocytes,  and 
also  in  the  overlying  fluid  from  such  an  exudate  after  the  cells  have 
been  sedimented  by  centrifugation. 

3.  Prolonged  centrifugation  of  serum  does  not  free  the  over- 
Ijdng  portion  from  the  virus. 

4.  It  is  impossible  to  free  the  blood  cells  in  defibrinated  blood 
from  the  virus  by  ten  to  twelve  washings  with  physiologic  salt 
solution,  although  this  process  decreases  the  virulence  of  the  blood. 
During  the  washing  a  point  is  reached  at  which  the  fluid  used  in 
washing  no  longer  contains  the  virus  in  infective  quantities. 

It  was  hoped  that  experiments  of  this  nature  might  bring  out 
evidence  in  favor  of  or  against  the  piroplasma  theory  of  Wilson 
and    Chowning.      I   beheve,   however,    that   the   results   do   not 

'Ibid.,  1906,  47,  p.  1067.     (See  p.  291  of  this  volume.) 

'  Ricketts,  Jour.  Infect.  Dis.,  1907,  4,  p.  141.     (See  p.  299  of  this  volume.) 


336  Contributions  to  Medical  Science 

justify  positive  conclusions  for  or  against  this  theory.  In  the 
experiments  cited  above  the  cell-free  serum  proved  rather  highly 
infective;  this  event,  however,  does  not  preclude  the  possibility 
of  a  piroplasmatic  infection,  for,  although  piroplasmas  invade  the 
erythrocytes  extensively  in  all  known  piroplasmic  diseases,  it  is 
self-evident  that  the  plasma  must  be  the  medium  through  which 
the  organisms  reach  the  erythrocytes.  Hence  in  all  piroplasmoses 
there  must  be  many  extracellular  organisms  at  some  stage  of  the 
disease. 

Similarly  the  result  quoted  above  in  paragraph  2  does  not  of 
necessity  disqualify  the  piroplasma  theory,  nor  does  it  support 
strongly  the  possibility  of  a  predominant  leukocytic  invasion  by 
the  parasite. 

The  results  stated  in  paragraph  4  would  seem  to  point  rather 
significantly  to  an  intimate  relation  between  the  virus  and  the 
red  or  white  cells.  However,  this  does  not  of  necessity  imply  an 
intracellular  location  of  the  parasite,  for  it  is  conceivable  that  the 
microbes  may  adhere  to  the  surface  of  the  blood  cells  either  through 
mutual  viscosity  or  some  other  attractive  or  adhesive  force.  I 
obtained  no  positive  result  in  attempting  to  liberate  the  organism 
from  its  hypothetical  position  within  the  blood  cells,  by  crushing 
the  latter  in  a  porcelain  ball-mill.  This  procedure  decreased  the 
virulence  of  the  blood,  which  probably  was  due  to  the  crushing  of 
many  of  the  organisms  in  the  mill. 

In  brief,  this  method  of  experimentation  does  not  give  results 
which  speak  conclusively  for  or  against  the  intracellular  location 
of  the  parasite,  and  for  this  reason,  if  for  no  other,  the  experiments 
may  have  some  worth. 

That  the  microbe  is  either  very  small  or  of  low  specific  gravity 
is  suggested  by  the  result  stated  above  in  paragraph  3. 

I  have  made  many  attempts  to  pass  the  virus  as  it  exists  in 
serum  through  the  porous  Berkefeld  candle  filters,  the  serum 
being  diluted  as  much  as  ten-fold  with  physiologic  salt  solution. 
In  no  instance  has  the  virus  passed  through  the  filters,  at  any 
rate  in  infective  quantities,  although  nine  cubic  centimeters  of 
serum  were  used  in  one  experiment.  The  failure  of  such  a  large 
quantity  of  filtered  serum  to  produce  infection,  when  it  is  known 


Transmission  of  Spotted  Fever  337 

that  the  unfiltered  serum  contains  the  virus  and  that  fresh  diseased 
blood  is  pathogenic  in  doses  of  0.05  to  o.  i  c.c,  is  significant.  The 
result  suggests  that  the  organism,  though  undoubtedly  minute, 
is  of  such  size  that  it  should  be  recognized  by  the  use  of  high  magni- 
fications, or  that  it  is  of  peculiar  form  or  possesses  such  adhesive 
properties  that  it  is  not  readily  filterable. 

Experiments  on  the  resistance  of  the  virus  to  cold,  heat,  desic- 
cation, dialysis,  and  other  agents  have  as  yet  furnished  no  dif- 
ferential clue  as  to  the  protozoan  or  bacterial  character  of  the 
microbe. 

That  recovery  from  one  attack  of  spotted  fever  renders  an  animal 
resistant  to  further  attempts  at  inoculation  is  one  of  the  important 
practical  facts  brought  out  by  my  experiments.  Studies  in  im- 
munity have  shown  that  a  disease  which  will  incite  immunity  in 
one  species  is  very  likely  to  have  a  similar  efifect  on  other  susceptible 
species,  hence  it  is  extremely  probable  that  man  generates  an 
immunity  similar  to  that  of  the  guinea-pig  which  has  recovered. 
This  supposition  is  strengthened  from  the  fact  that  no  authentic 
instance  of  two  attacks  in  man  has  been  observed  in  so  far  as 
one  can  learn. 

It  is,  then,  somewhat  reasonable  to  hope  that  an  efficient 
vaccine  or  curative  serum  may  be  prepared  provided  the  micro- 
organism lends  itself  to  suitable  manipulations.  I  have  certain 
guinea-pigs  which  are  called  hyperimmune.  Since  their  recovery 
from  spotted  fever  they  have  received  many  injections  of  infected 
blood.  Assisted  by  Mr.  Gomez,  it  has  been  possible  to  show 
recently  that  o.i  to  0.3  c.c.  of  hyperimmune  blood  will  protect  a 
guinea-pig  against  twenty  or  more  minimum  pathogenic  doses  of 
the  diseased  blood.     These  investigations  are  being  continued.* 

It  is  most  interesting  that  the  offspring  of  an  immune  female 
guinea-pig  also  are  immune  for  a  period,  the  limits  of  which  have 
not  yet  been  determined.' 

By  showing  the  transmissibility  of  spotted  fever  to  the  guinea- 
pig  and  monkey,  a  means  of  putting  the  tick  theory  to  a  decisive 
test  had  been  provided. 

In  attacking  this  problem  the  first  logical  step  was  to  determine 

«  Not  heretofore  published. 


338  Contributions  to  Medical  Science 

whether  the  tick  after  feeding  on  a  diseased  animal  is  able  to  infect 
a  healthy  animal  by  biting  it.  If  this  should  prove  to  be  true,  and 
if,  in  addition,  it  could  be  shown  that  the  relation  of  the  virus  to 
the  tick  is  a  very  intimate  one,  so  that  the  transmission  would 
appear  to  be  "biological"  rather  than  purely  mechanical,  the 
results  would  place  the  tick  under  the  gravest  suspicion  as  the 
means  of  infecting  man. 

In  the  summer  of  1906  I  showed  that  the  female  tick  can  acquire 
and  transmit  spotted  fever  by  means  of  biting  (see  p.  278  of  this 
volimie),^  and  King^  of  the  Hygienic  Laboratory  obtained  a 
similar  result  at  the  same  time  and  with  the  same  strain  (my 
strain)  of  the  virus. ^  Later  in  the  year  I  found  that  the  male 
tick  also  may  acquire  and  transmit  the  disease,  which  could  well  be 
anticipated  since  the  male  as  well  as  the  female  is  a  blood-sucker. 

Further  experiments  have  shown  the  existence  of  a  very  intimate 
relationship  of  the  virus  to  the  tick  and  that  the  transmission 
must  be  regarded  as  biological  in  character  rather  than  mechanical 
throughout.  In  no  instance  has  a  tick  ceased  to  be  infective 
so  long  as  it  lived  and  would  feed,  a  period  of  several  months.  It 
may  acquire  the  disease  as  a  larva  or  as  a  nymph,  retain  it  during 
moulting,  and  prove  infective  when  it  reaches  the  subsequent 
active  stage  (see  pp.  312  and  324  of  this  volume).'' 

Of  great  importance  is  the  hereditary  transmission  of  the 
disease  by  an  infected  female.  This  occurred  in  two  of  my  "hered- 
ity experiments,"  and  this  may  have  an  important  bearing  on 
the  vitality  of  the  disease  in  nature  from  year  to  year.  The  term 
"hereditary"  is  used  rather  broadly  and  only  in  the  sense  that 

'  Ricketts,  Jour.  Amer.  Med.  Assn.,  1906,  47,  p.  358. 

'  King,  Pub.  Health  Rep.,  1906. 

'  At  the  time  this  work  was  done  I  had  no  knowledge  of  previous  experiments  which  demonstrated 
the  ability  of  the  "wood-tick"  to  transfer  spotted  fever.  Recently  I  have  had  authoritative  information 
that  Dr.  L.  P.  McCalla  and  Dr.  H.  A.  Brereton,  of  Boise  City,  Idaho,  transmitted  the  disease  from  man  to 
man  in  two  experiments.  The  tick  was  obtained  "from  the  chest  of  a  man  very  ill  with  spotted  fever," 
and  "applied  to  the  arm  of  a  man  who  had  been  in  the  hospital  for  two  months  and  a  half,  and  had  lost 
both  feet  from  gangrene  due  to  freezing."  On  the  eighth  day  the  patient  became  ill  and  passed  through  a 
mild  course  of  spotted  fever,  leaving  a  characteristic  eruption.  The  experiment  was  repeated  by  placing 
the  tick  on  a  woman's  leg,  and  she  likew^ise  was  infected  with  spotted  fever.  Although  these  results  re- 
ceived no  publicity  other  than  that  given  in  reports  to  local  societies,  I  take  pleasure  in  according  to  Dr. 
McCalla  and  to  his  colleague,  Dr.  Brereton,  the  credit  of  having  first  shown  that  the  tick  may  act  as  a 
carrier  of  spotted  fever.  Their  experiments  preceded  by  more  than  a  year  those  of  Dr.  King  and  of  myself, 
and  they  were  pyerformed  with  the  full  consent  of  the  patients  concerned. 

<  Ricketts,  Jour.  Am.  Med.  Assn.,  1907,  49,  p.  24;  ibid.,  p.  1278. 


Transmission  of  Spotted  Fever  339 

the  infection  was  derived  from  the  female.  It  may  be  argued 
that  during  the  process  of  oviposition,  in  which  the  head  parts 
play  a  function,  virus  may  have  been  deposited  on  the  surface 
of  the  eggs  from  the  saUvary  glands  of  the  female  and  that  the 
microbes  reached  the  embryo  or  its  food  supply  by  penetrating 
the  egg  membrane,  or  that  the  virus  having  been  deposited  on 
the  surface  of  the  egg,  the  embryo  later  became  infected  by  con- 
suming a  portion  of  the  membrane.  The  pertinence  of  the  first 
argument  must  be  recognized,  although  the  virus  has  shown  no 
penetrating  power  for  the  skin  of  man  or  for  the  mucous  membranes 
of  man  and  animals,  membranes  which  would  seem  to  be  much 
more  susceptible  to  penetration  than  the  dense  covering  of  the 
tick's  egg.  The  second  argument  could  not  be  considered  as  valid 
because  the  life  of  the  virus  when  exposed  to  the  light  is  known 
to  be  much  shorter  than  the  time  required  for  the  development 
of  the  embryo.  Such  considerations,  however,  do  not  aflfect  the 
practical  fact  that  the  larvae  of  infected  females  may  be  born  with 
the  virus  of  spotted  fever  in  their  saHvary  glands,  and  that  they 
are  sometimes  able  to  transmit  the  disease  to  a  susceptible  animal. 

"Hereditary"  transmission  was  attained  in  only  two  out  of 
twenty-six  experiments,  a  fact  which  fits  well  with  the  scarcity 
of  infected  ticks  in  nature  (see  below)  and  in  the  rarity  of  the 
disease  in  man. 

I  am  inclined  to  the  view  that  the  tick  during  the  time  it  harbors 
the  virus  undergoes  a  generalized  infection.  I  take  it  that  the 
successful  "heredity  experiments"  signify  uterine,  hence  general- 
ized infection.  I  have  shown  by  inoculation  experiments  that  the 
virus  is  present  in  the  alimentary  sac  and  in  the  salivary  glands  of 
the  infected  adult  tick.  Undoubtedly  the  ahmentary  sac  becomes 
infected  primarily  through  the  diseased  blood  ingested  by  the  tick, 
and  it  is  probable  that  the  localization  of  the  virus  in  the  salivary 
glands  is  a  part  of,  or  residuum  of,  a  generalized  infection. 

The  second,  and  one  may  say  the  final,  step  required  to  prove 
that  the  tick  is  the  agent  through  which  man  is  infected,  involved 
the  discovery  of  infected  ticks  in  nature.  I  say  the  final  step, 
because  it  would  complete  a  chain  of  the  strongest  possible  circum- 
stantial evidence  tending  to  incriminate  the  tick. 


340  Contributions  to  Medical  Science 

On  account  of  the  disproportion  between  the  number  of  tick- 
bitten  persons  and  the  number  of  cases  of  spotted  fever  occurring 
any  particular  season,  it  was  anticipated  that  the  search  would  be 
a  di£5cult  task  and,  with  negative  results,  might  require  the  testing 
of  many  thousand  ticks  before  one  would  be  justified  in  acknowl- 
edging failure. 

In  order  to  increase  the  probability  of  finding  infected  ticks 
in  nature  those  selected  for  the  tests  were  taken  from  localities 
known  to  be  infected,  some  being  obtained  from  horses  and  cattle 
and  others  from  the  clothing  of  different  individuals  who  had 
collected  them  in  this  way  from  the  woods.  They  were  allowed 
to  feed  in  groups  on  normal  guinea-pigs,  and  for  a  period  which 
is  longer  than  that  required  for  transmission. 

Five  hundred  and  thirteen  ticks  were  tested  in  this  way  and  of 
this  number  two  hundred  and  ninety-six  were  found  attached  to 
the  guinea-pigs.  It  is  quite  certain  that  this  does  not  represent 
the  full  number  which  fed,  since  in  searching  the  skin  of  the  guinea- 
pig  for  ticks  a  few  may  unavoidably  be  overlooked. 

Eventually  one  or  more  infected  ticks  were  found  among  a 
group  of  thirty-six  males  which  had  been  collected  from  apparently 
healthy  horses.  The  guinea-pig  on  which  they  fed  developed 
fever  on  the  seventh  day  after  being  placed  with  the  ticks  and  died 
seven  days  later,  exhibiting  the  clinical  and  anatomic  changes 
which  render  spotted  fever  easily  recognizable  in  this  animal. 
No  micro-organism  could  be  cultivated  from  the  blood  or  organs 
of  the  animal,  which,  as  stated  above,  is  the  customary  result 
of  cultivation  experiments.  Seventeen  ticks  were  seen  attached 
to  the  guinea-pig,  the  first  being  discovered  on  the  second  day. 

The  experiment  was  carried  further  by  producing  the  disease 
in  another  guinea-pig  through  the  injection  of  blood  from  the  first 
and  in  a  monkey  by  a  transfer  from  the  second  guinea-pig.  In 
addition,  twenty-four  of  the  ticks  which  had  been  placed  with  the 
first  guinea-pig  two  days  before  the  onset  of  fever  were  given  a 
second  test  in  two  groups  of  twelve  each.  One  of  the  two  guinea- 
pigs  died  of  peritonitis  after  four  days.  The  other  began  a  febrile 
course  on  the  sixth  day  and  was  found  dead  five  days  later  with 
anatomic  changes  and  cultural  results  which  rendered  the  diagnosis 


Transmission  of  Spotted  Fever  341 

of  spotted  fever  positive.  Six  ticks  were  seen  attached  to  this 
animal.  The  experiment  was  not  carried  further,  since  the  ticks 
were  still  feeding  when  fever  developed  and  all  had  had  the  oppor- 
tunity of  becoming  infected. 

No  precaution  was  neglected  to  place  these  experiments  above 
criticism.  Each  animal  with  its  ticks  was  isolated  in  a  tick-proof 
cage  in  all  instances,  and  the  cages  either  had  never  been  used 
before  or,  if  used,  had  been  sterilized  before  being  subjected  to  use 
again.     No  tick  of  uncertain  identity  was  utilized. 

The  finding  of  infected  ticks  on  the  horse  does  not  necessarily 
implicate  the  horse  as  a  factor  in  the  maintenance  of  the  disease 
in  nature.  In  so  far  as  I  can  judge  by  preliminary  experiments, 
this  animal  is  not  very  susceptible  to  spotted  fever. 

I  have  accordingly  shown  the  following  points  concerning 
the  relationship  of  the  wood-tick  to  Rocky  Mountain  spotted 
fever : 

1.  The  disease  may  be  acquired  and  transmitted  by  the  larva, 
the  nymph,  and  the  adult  male  and  female,  that  is  to  say,  by  the 
tick  during  any  of  its  active  stages. 

2.  The  larvae  of  an  infected  female  are  in  some  instances 
infective. 

3.  The  association  of  the  virus  with  the  tick  is  very  intimate,  as 
shown  by  the  long  duration  of  infectivity  of  the  tick,  the  retention 
of  infectivity  during  moulting,  and  the  "hereditary"  trans- 
mission of  the  virus;  hence  there  is  some  reason  to  believe  that  the 
tick  suffers  from  a  relatively  harmless  generalized  infection,  and 
that  the  virus  proliferates  in  its  body.  The  disease  probably  is 
transferred  through  the  salivary  secretion  of  the  tick,  since  the 
salivary  glands  of  the  infected  adult  contain  the  virus.  There 
seems  to  be  no  doubt  but  that  the  arachnid,  when  biting,  injects 
some  of  the  salivary  secretion  into  the  skin;  this  is  indicated  by 
the  marked  local  reaction  which  follows  the  bite  of  even  the  normal 
tick. 

4.  Infected  ticks  exist  in  nature  in  small  numbers  in  the  so- 
called  infected  districts. 

When  one  adds  to  such  data  the  generally  known  facts  that  the 
disease  in  man  is  limited  to  the  season  of  prevalence  of  the  adult 


342  Contributions  to  Medical  Science 

tick,  the  larvae  and  nymphs  rarely  feeding  on  man,  and  that  a 
history  of  recent  tick  bite  may  be  obtained  in  nearly  every  case 
of  spotted  fever,  and  cannot  be  positively  excluded  in  the  event 
that  such  history  is  not  given,  it  seems  necessary  to  conclude 
that  the  chain  of  circumstantial  evidence  is  now  complete,  and  that 
the  acceptance  of  the  theory  of  transmission  by  the  bite  of  the 
tick  is  now  justified. 

I  have  failed,  and  Mr.  P.  G.  Heinemann  who  assisted  in  the 
work  this  year  Hkewise  failed,  to  cultivate  or  identify  positively 
the  micro-organism.  It  has  not  been  possible  so  far  to  identify 
or  accept  the  piroplasma  of  Wilson  and  Chowning  as  the  cause  of 
the  disease. 

Concerning  the  gopher  hypothesis,  occasional  susceptible 
animals  are  found  among  the  local  species,  but  it  is  not  certain 
that  the  gopher  plays  the  part  in  the  perpetuation  of  the  disease 
which  Wilson  and  Chowning  assumed  that  it  plays. 

The  way  is  now  open  to  carry  on  an  intelligent  campaign 
against  the  disease  by  destroying  massive  numbers  of  the  ticks, 
through  the  use  of  methods  which  have  been  so  successful  in  the 
fight  against  Texas  fever  of  cattle  in  the  southern  states.* 

'  Since  this  article  was  written  the  search  for  a  vaccine  has  progressed  so  favorably  that  there  is 
reason  to  believe  that  vaccination  will  prove  an  efEective  means  of  preventing  the  disease  in  man.  This 
work  will  be  reported  in  detail  in  the  near  future. 


STUDIES  ON  IMMUNITY  IN  ROCKY  MOUNTAIN 
SPOTTED  FEVER.^ 

H.     T.     RiCKETTS     AND    L.     G  O  M  E  Z. 
{From  the  Departments  of  Pathology  and  Experimental  Therapy,  The  University  of  Chicago.) 

In  these  studies  it  has  been  our  aim  to  ascertain  with  certainty- 
some  of  the  fundamental  facts  concerning  immunity  to  this  disease 
and  to  pass  as  rapidly  as  possible  to  the  practical  aspects  of  the 
question,  namely,  the  study  of  serum  prevention,  serum  therapy, 
and  specific  vaccination.  Concerning  these  three  important 
phases  of  the  work,  we  report  preliminary  experiments  which 
indicate  the  scope  of  our  studies,  the  results  of  which  will  be  reported 
more  exhaustively  in  the  future. 

Diagnosis  of  the  Experimental  Disease. 

Before  considering  the  experiments  it  seems  desirable  to  point  out  the  essential 
clinical  and  anatomical  features  of  experimental  spotted  fever  in  order  to  show  that 
the  criteria  for  the  recognition  and  differentiation  of  the  disease  are  of  a  definite  and 
convincing  character.  While  diagnosis  is  usually  easy  concerning  infections  caused 
by  organisms  that  may  be  cultivated  and  that  show  definite  biologic  characteristics, 
or  that  have  a  definite  morphology,  as  in  the  case  of  certain  protozoa,  it  would  appear 
to  be  more  difficult  and  liable  to  error  wnen  the  organism  is  unrecognized  and  uncul- 
tivated. 

The  incubation  period  is  definite  and  is  never  absent  in  animals  that  were  fever 
free  and  that  suffered  an  accident  at  the  time  of  inoculation  such  as  might  be  caused 
by  puncture  of  the  intestines  or  some  accidental  infection.  Following  intraperitoneal 
inoculation  in  the  guinea-pig,  monkey,  or  rabbit,'  two  to  four  or  five  days  elapse 
before  a  distinct  rise  in  the  temperature  occurs.  The  incubation  period  is  from  two 
to  four  days  longer  when  the  inoculation  is  subcutaneous.  Its  length  also  has  a  cer- 
tain relation  to  the  quantity  of  virus  inoculated;  when  the  minimum  pathogenic 
dose  is  used  it  is  often  from  one  to  three  days  longer  than  when  several  multiples  of 
this  dose  are  injected. 

At  the  close  of  the  incubation  period  the  temperature  rises  to  a  maximum  rapidly. 
Not  infrequently  it  is  normal  (about  102 . 5)  on  one  day,  and  on  the  next  day,  105  or 

»  From  Jour.  Infect.  Dis.,  1908,  5,  p.  221.  The  essential  points  of  this  paper  were  presented  before 
the  Section  in  Pathology  and  Physiology  of  the  American  Association  for  the  Advancement  of  Science, 
January  2,  1908.  This  work  has  been  supported  largely  by  the  Department  of  Experimental  Therapy, 
The  University  of  Chicago,  through  the  kindness  of  Professor  S.  A.  Matthews,  although  the  appropriation 
of  the  Department  of  Pathology  and  funds  provided  by  the  state  of  Montana  have  been  drawn  on  to 
a  certain  extent. 

'  The  rabbit  has  recently  been  found  to  be  susceptible  to  the  strains  now  cultivated  in  laboratory 
animab.    (See  Gomez,  "  Rocky  Mountain  Spotted  Fever  in  the  Rabbit,"  Jour.  Infect.  Dis.,  1909,  6,  p.  383.) 

343 


344  Contributions  to  Medical  Science 

even  io6.'  More  often,  however,  the  first  day  of  the  febrile  period  is  marked  by  a 
temperature  of  103  to  104  with  a  more  pronounced  rise  on  the  second  and  following 
days.  The  course  is  that  of  a  continuous  fever  throughout,  as  in  man.  For  working 
purposes  it  is  satisfactory  to  take  the  temperature  but  once  a  day  and  it  is  immaterial 
whether  this  is  done  in  the  morning  or  afternoon.  Sometimes  the  maximum  tem- 
perature is  not  reached  until  the  third  or  fourth  day  of  fever.  Usually  the  high  point 
is  about  106,  although  a  temperature  of  107 . 2  has  been  observed. 

In  fatal  cases  the  duration  of  the  fever  is  subject  to  variations,  some  dying  after 
four  or  five  days  and  others  after  eight  or  ten  days  of  fever.  Similarly  animals  which 
recover  may  exhibit  fever  for  from  six  to  ten  days.  In  recovery  subsidence  of  the 
temperature  is  usually  gradual.  Occasionally,  however,  a  drop  to  normal  is  noted 
within  24  hours,  and  it  may  be  shghtly  below  normal  for  one  or  two  days.  In  fatal 
cases  the  reduction  is  sudden,  and  a  drop  to  97  or  99  is  an  unfaiUng  sign  of  impending 
death. 

The  most  characteristic  external  signs  of  the  disease  are  emaciation,  a  roseolar 
eruption,  hemorrhages  into,  and  gangrene  of,  the  external  genitalia.  The  roseolar 
eruption  begins  on  the  third  to  the  fifth  day  of  fever  and  is  best  seen  on  the  external 
genitalia  of  guinea-pigs  with  white  skins.  If  such  animals  are  shaved  it  may  also 
be  detected  on  other  parts  of  the  body,  especially  the  thighs,  back,  and  face.  It  is  not 
always  recognizable  and  escapes  observation  entirely  on  animals  having  dark  skins. 
In  males  the  scrotum  begins  to  swell  after  from  two  to  four  days  of  fever  and  it  is 
characteristic  for  the  skin  at  this  point  to  become  densely  infiltrated  with  blood; 
in  the  event  that  the  animal  lives  long  enough,  i.e.,  in  the  event  of  recovery,  the  hem- 
orrhagic areas  become  gangrenous  and  separate,  leaving  greater  or  less  deformity  of 
the  scrotum.  These  phenomena  are  not  absolutely  constant  but  occur  in  a  high 
percentage  of  the  males.  Apparently  the  swelling  of  the  scrotum  is  due  chiefly  to 
congestion  and  edema  of  the  underlying  connective  tissue,  the  tunica  vaginalis  and 
the  epididymis.  In  females,  the  vulva  becomes  swollen  and  may  show  the  roseolar 
eruption  as  stated;  hemorrhage  and  gangrene  of  the  vulva,  however,  are  rather  un- 
common. Frequently  the  ears  suffer  from  gangrene  to  a  greater  or  less  extent  and 
it  is  the  impression  that  this  phenomenon  now  occurs  more  frequently  than  when 
the  virus  was  first  cultivated  from  man.  Occasionally  both  ears  fall  away  to  their 
bases.  Less  frequently  the  soles  of  the  feet  undergo  gangrene.  In  fatal  cases  the 
g\iinea-pig  loses  approximately  one-third  of  its  weight  during  the  disease. 

The  most  characteristic  findings  at  autopsy,  aside  from  the  changes  in  the  geni- 
talia, are  a  greatly  enlarged  spleen  and  enlarged  and  hemorrhagic  lymph  glands  with 
extreme  congestion  of  the  adjacent  areolar  tissue.  Enlargement  of  the  suprarenal 
glands  is  of  frequent  occurrence  and  they  sometimes  contain  small  hemorrhages. 

A  valuable  point  for  the  corroboration  of  the  diagnosis  is  the  failure  to  cultivate 
any  micro-organism  from  the  blood  or  organs  of  animals  that  have  been  killed  during 

'  It  has  been  the  rule  in  taking  the  temperature  of  guinea-pigs  to  insert  the  thermometer  as  far  as 
possible  into  the  colon.  If  this  distance  is  not  more  than  two  inches,  it  is  usually  because  the  colon  is 
occluded  by  feces,  and  this  condition  is  recorded  at  such  times.  In  animals  of  300  to  400  grams  weight 
it  is  usually  possible  to  reach  a  depth  of  about  three  inches,  and  in  full  grown  guinea-pigs  the  thermome- 
ter can  be  inserted  for  almost  its  entire  length.  (This  is  the  ordinary  clinical  thermometer.)  A  large 
number  of  observations  indicates  that  the  normal  temperature  of  the  guinea-pig,  as  taken  in  this  way,  lies 
between  102.2  and  102.6.  A  temperature  above  103  would  seem  to  be  abnormal,  although  occasional 
groups  of  animals  are  encountered  in  which  the  temperatures  may  vary  between  103  and  104.5,  without 
any  evident  cause.  This  may  persist  for  several  days  and,  naturally,  causes  confusion  in  the  recognition 
of  spotted  fever. 


Immunity  in  Spotted  Fever  345 

the  course  of  the  fever,  or  which  have  died  as  a  natural  result  of  the  infection.  This 
statement  refers  to  typical  cases,  in  which  the  clinical  course  indicates  that  there  was 
no  mixed  infection,  and  in  the  case  of  autopsies  it  applies  to  those  that  are  performed 
immediately  or  soon  after  death. 

This  negative  bacteriologic  finding  has  been  constant  through  two  years  of  work 
and  although  it  is  of  the  greatest  importance  as  a  diagnostic  point,  the  clinical  course 
of  the  disease  is  so  characteristic  that  cultures  are  now  resorted  to  only  occasionally 
to  prove  the  purity  of  the  virus  used  for  passage,  and  in  experiments  to  serve  as  a 
check  for  animals  in  which  there  has  been  some  departure  from  the  normal  course 
of  the  disease  or  in  which  the  autopsy  reveals  unusual  conditions.  If  the  animals 
used  for  an  experiment  are  free  from  fever  when  the  experiment  is  begun,  and  if  fol- 
lowing injection  they  or  their  controls  exhibit  the  usual  incubation  period  followed 
by  a  typical  course  of  fever  and  the  development  of  the  cutaneous  phenomena,  and 
if  the  findings  at  autopsy  are  as  described  above  the  experiments  are  sufficiently 
controlled  for  working  purposes.  This  method  has  been  found  perfectly  satisfactory 
and  in  two  instances  was  sufficient  for  the  recognition  of  adventitious  epidemics,  the 
presence  of  which  was  subsequently  verified  by  bacteriologic  methods. 

In  view  of  the  fact,  as  stated  later,  that  an  attack  of  spotted  fever,  however 
mild,  confers  strong  immunity,  an  "immunity  test"  is  of  value  in  determining  whether 
animals  which  recover  have  or  have  not  suffered  from  spotted  fever.  Our  experience 
indicates  that  any  used  animal  which  develops  spotted  fever  after  the  injection  of 
20  to  50  multiples  of  the  minimum  pathogenic  dose  of  virus  could  not  have  had  the 
disease  in  the  first  instance. 

The  Virus. 

For  the  major  part  of  the  work  two  strains  of  the  virus  have  been  used,  one 
obtained  in  the  spring  of  1906  and  the  other  in  1907.  The  first  is  known  as  the  Bradley 
strain,  the  second  as  the  Eddy  strain.  During  the  summer  of  1906  the  Bradley 
strain  was  grown  alternately  in  monkeys  and  guinea-pigs  and  subsequently  in  guinea- 
pigs  alone.  The  Eddy  strain  has  been  cultivated  in  guinea-pigs  exclusively.  The 
method  of  cultivation  is  that  of  the  ordinary  passage,  the  blood  of  an  infected  animal 
being  injected  intraperitoneally  or  subcutaneously  into  a  healthy  one.  The  Bradley 
strain  has  undergone  approximately  125  passages.  The  two  strains  show  about 
the  same  degree  of  virulence  judging  from  the  quantity  of  virulent  blood  required 
to  produce  infection.  On  this  account  they  have  been  used  more  or  less  interchange- 
ably, a  record  being  kept  of  the  strain  used  for  each  experiment. 

It  is  important  to  know  whether  the  virus  used  in  these  experiments  is  the  same 
specifically,  as  when  first  obtained.  We  have  the  following  evidence  that  it  is:  Ani- 
mals that  had  recovered  from  the  infection  with  the  virus  after  it  had  been  culti- 
vated in  the  guinea-pig  for  one  year  were  immune  to  the  inoculation  of  fresh  virus 
obtained  from  man  in  the  spring  of  1907.  Of  two  strains,  one  obtained  in  1906  and 
one  in  1907,  each  confers  immunity  against  the  other.  This,  of  course,  is  independent 
of  the  possibility  that  the  virus  has  undergone  variations  in  the  character  of  its  patho- 
genicity as  a  result  of  prolonged  cultivation  in  the  guinea-pig. 

The  defibrinated  blood  of  infected  animals,  the  blood  being  drawn  from  the 
carotid  artery  or  the  heart,  is  the  source  of  the  virus,  and  when  the  term  "virus"  is 
used  it  is  understood  that  it  refers  to  such  blood.  The  virus  has  been  standardized 
as  accurately  as  possible  in  terms  of  the  minimum  pathogenic  dose.     It  is  impossible 


346  Contributions  to  Medical  Science 

to  deal  with  the  minimum  lethal  dose  because  of  the  fact  that  minimum  infective 
doses  are  about  as  likely  to  prove  fatal  as  several  multiples  of  such  doses.  In  most 
instances  the  minimum  pathogenic  dose  lies  between  o.oi  c.c.  and  0.03  c.c.  The 
former  frequently  fails  to  produce  infection  whereas  the  latter  is  nearly  always  infect- 
ive. For  quantitative  work  it  is  important  to  draw  the  virus  with  respect  to  the 
duration  of  the  infection,  because  of  the  possibility  that  antibodies  which  may  appear 
later  in  the  course  may  lessen  infectivity.  As  illustrating  the  importance  of  this 
consideration,  two.  strains  were  lost  in  the  spring  of  1906  by  waiting  until  the  animals 
had  died  before  attempting  to  perpetuate  the  disease  in  succeeding  generations. 

We  have  gained  the  impression  that  virus  which  is  taken  rather  late  in  the  disease 
is  more  likely  to  produce  infection,  if  it  is  diluted  several  fold  with  salt  solution. 

It  has  happened  a  number  of  times  that  small  doses  were  infective  whereas  larger 
doses  of  the  same  virus  were  not.  Such  an  experience  occurred  recently  when  infected 
blood  from  a  horse,  drawn  rather  late  in  the  disease,  was  injected  into  two  guinea- 
pigs  in  doses  of  i  c.c.  and  5  c.c;  the  former  amount  caused  spotted  fever  whereas  the 
latter  did  not.  This  was  not  due  to  lack  of  susceptibility  on  the  part  of  the  latter 
animal,  inasmuch  as  it  contracted  the  disease  later  when  inoculated  with  virus  from 
the  guinea-pig.  We  are  inclined  to  the  view  that  the  failure  of  large  doses  to  infect 
under  these  circumstances  is  due  to  the  comparatively  large  quantity  of  antibodies 
in  the  larger  quantities  of  blood.  It  is  true  that  the  proportionate  quantity  of  anti- 
bodies to  the  number  of  micro-organisms  in  5  c.c.  is  the  same  as  in  i  c.c.  of  blood,  yet 
when  the  smaller  quantity  is  used  the  antibodies  are  subject  to  greater  dilution  and 
it  is  in  accordance  with  the  known  action  of  other  antibodies  to  assume  that  their 
effect  on  the  micro-organisms  would  be  somewhat  attenuated  in  this  instance  by  their 
dilution.  Also  there  are  good  general  grounds  for  believing  that  antibodies  may  be 
fixed  or  destroyed  by  the  tissues  to  a  certain  extent,  and  if  this  is  true  the  residuum  of 
effective  antibodies,  in  proportion  to  the  number  of  micro-organisms,  would  be  lower 
when  small  quantities  of  virus  are  introduced  than  when  larger  quantities  are  given. 

For  most  purposes  the  injections  have  been  intraperitoneal,  although  the  subcu- 
taneous route  has  been  employed  for  certain  e.xperiments. 

CONCERNING  IMMUNITY   IN   MAN. 

In  SO  far  as  we  have  been  able  to  learn,  there  are  no  data  regard- 
ing the  immunity  of  man  to  Rocky  Mountain  spotted  fever, 
although  our  knowledge  of  the  matter  is  limited  to  the  conditions 
in  the  Bitter  Root  Valley  in  Montana.  There  is  no  authoritative 
example  of  two  attacks  of  the  disease  in  the  same  person  in  this 
locahty.  It  is  true  that  one  or  more  individuals  state  their  behef 
that  they  have  had  more  than  one  attack,  but  such  statements 
lack  the  confirmation  of  the  local  physicians  who  are  experienced 
in  the  recognition  of  the  disease. 

The  results  obtained  in  the  monkey  and  the  guinea-pig  suggest 
that  man  probably  acquires  a  strong  immunity  as  a  result  of  an 
attack.     This  is   a  question,  however,  which  can  be  approached 


Immunity  in  Spotted  Fever  347 

from  the  experimental  side  by  a  study  of  the  serum  of  those  who 
have  recovered. 

ACTIVE   IMMUNITY   IN  THE   MONKEY. 

The  fact  that  one  attack  of  spotted  fever  renders  the  monkey 
immune  to  a  second  inoculation,  performed  some  months  later, 
has  aheady  been  reported  by  one  of  us  (see  p.  299  of  this  volume).'' 
An  additional  experiment  illustrating  the  same  point  may  be  cited 
briefly. 

On  May  20,  1906,  the  monkey,  a  large  M.  rhesus,  received 
intraperitoneally  10  c.c.  of  defibrinated  blood  from  the  patient. 
Porter.  After  an  incubation  period  of  about  two  days  its  tempera- 
ture rose  rapidly  to  105.3  ^^  the  vicinity  of  which  it  remained  for 
three  days,  and  then  gradually  returned  to  normal  during  the  course 
of  five  days  more.  The  animal  showed  no  eruption,  although  his 
eyes  became  much  reddened.  His  blood  was  not  inoculated  into 
other  animals  in  order  to  confirm  the  diagnosis  of  spotted  fever 
and  on  this  account  there  naturally  existed  a  good  deal  of  doubt  as 
to  whether  the  monkey  had  been  infected  with  spotted  fever  by 
the  inoculation. 

On  February  i,  1907,  the  monkey  was  given  an  immunity 
test,  a  control  monkey  (No.  24)  being  inoculated  with  the  same 
quantity  of  virus.  The  inoculation  caused  no  rise  in  the  tempera- 
ture of  the  animal,  whereas  the  temperature  of  the  control,  after 
a  brief  incubation  period,  rapidly  rose  to  106.  i,  near  which  it 
remained  until  the  animal  was  killed,  on  the  sixth  day  after  inocula- 
tion.^ On  the  second  day  of  high  fever  the  control  presented 
a  roseolar  eruption  of  the  skin  of  the  perineum,  which  was  hemor- 
rhagic at  the  time  it  was  killed.  The  blood  of  this  animal  pro- 
duced spotted  fever  in  guinea-pigs. 

The  experiment  reported  previously  and  the  one  just  cited 
indicate  that  an  attack  of  spotted  fever  confers  immunity  in  the 
monkey,  which  in  the  last  instance  was  present  in  a  marked  degree 
for  nine  months.  The  quantity  of  virus  injected  was  very  large, 
consisting  of  defibrinated  blood  and  a  dense  emulsion  of  the  liver, 

■  Ricketts,  "Further  Observations  on  Rocky  Mountain  Spotted  Fever,  etc.,"  Jour.  Am.  Med. 
Assn.,  1906,  47,  p.  1067. 

'  It  was  the  custom  at  that  time  to  kill  the  monkeys  during  the  height  of  the  fever  in  order  to  obtain 
quantities  of  virus  for  further  experiments. 


348  Contributions  to  Medical  Science 

spleen,  and  kidneys  of  an  infected  guinea-pig,  a  total  quantity 
of    15  c.c. 

ACTIVE   IMMUNITY   IN   THE   GUINEA-PIG. 

In  a  previous  article  it  was  stated  that  the  guinea-pig  that  has 
recovered  from  spotted  fever  is  thereafter  immune  to  the  disease, 
although  the  details  of  experiments  were  not  given  at  that  time.' 
This  result  has  proved  to  be  a  rule  to  which  no  exception  has  been 
found  among  a  great  many  tests.  An  illustrative  experiment 
will  be  cited  in  this  place  and  other  instances  are  found  through 
the  remaining  part  of  this  report. 

Guinea-pig  710  was  inoculated  with  spotted  fever  by  larvae 
of  an  infected  tick.  Twelve  days  after  the  first  larvae  were  seen 
to  be  attached,  the  temperature  of  the  guinea-pig  rose  suddenly 
to  105.2,  and  on  successive  days  was  read  at  105.3,  104 -8,  104.3, 
103.8,  102.3,  ^^^  102.2.  This  was  a  short  course  of  fever  and 
some  doubt  existed  concerning  the  correctness  of  the  diagnosis 
until  the  result  of  a  transfer  inoculation  was  known.  On  the  day 
when  the  temperature  of  guinea-pig  710  registered  104.3,  3  c.c. 
of  blood  were  drawn  from  the  heart  and  injected  intraperitoneally 
into  guinea-pig  749.  The  latter  passed  through  a  typical  course 
of  spotted  fever,  the  temperature  being  as  follows  on  succeeding 

days  after  inoculation:    103.4,  103.4, ,  105.6,  104.8,  106.7, 

106.4,  106,  104.6,  105.4,  104.4,  io4j  I04-3)  io3>  10355  recovery. 
The  scrotum  of  749  became  hemorrhagic  and  sloughed  in  a  typical 
manner,  and  the  animal,  furthermore,  resisted  a  subsequent 
immunity  test.  After  these  experiments  had  proved  beyond 
doubt  that  guinea-pig  710  had  been  infected  by  the  larval  ticks, 
the  animal  was  given  an  immunity  test  by  the  intraperitoneal 
injection  of  2  c.c.  of  virus  from  guinea-pig  781,  which  had  been 
inoculated  with  the  Eddy  strain  of  the  virus.  On  successive 
days  the  temperature  of  guinea-pig  710  was  as  follows:  103.4, 
103,  102.6,  102.4,  102.3,  102.5,  102.7,  103. 1,  102.3, J  103.6. 

A  good  control  for  the  experiment  cited  is  found  in  the  history 
of  guinea-pig  705  which  also  was  used  to  determine  whether  or 
not  the  infected  female  tick  transmits  the  disease  to  her  progeny. 
Twenty-two  days  after  the  larvae  of  an  infected  tick  were  placed 

■  Ricketts,  loc.  cil. 


Immunity  in  Spotted  Fever  349 

with  the  animal,  the  latter  had  four  days  of  moderate  fever:  104. 3, 

104. 5,  103 . 1,  104. 1,  102 . 1.  This  event  suggested  that  the  guinea- 
pig  may  have  been  given  a  mild  infection  by  the  larvae,  but  this 
was  by  no  means  certain  because  the  animals,  confined  and  often 
exhausted  as  they  are  during  such  experiments,  sometimes  exhibit 
abnormal  temperatures  for  one  or  several  days  which  is  entirely 
independent  of  infection  with  spotted  fever.  About  three  months 
later  this  animal  received  an  injection  of  2.5-  c.c.  of  Bradley  virus 
with  the  following  result:  102,  102.5,  102.8,  105,  105.  i,  104.7, 
104.8,  103.9,  99.6;  death.  The  figures  represent  the  temperature 
of  the  animal  on  successive  days.  On  the  seventh  day  after 
inoculation  the  vulva  became  hemorrhagic;  the  autopsy  showed 
typical  changes  in  the  spleen  and  lymph  glands.  Cultures  were 
sterile. 

It  is  a  matter  of  first  importance  that  a  mild  attack  of  spotted 
fever  results  in  the  formation  of  strong  immunity.  Usually,  when 
small  doses  of  virus  are  given,  the  animals  either  react  with  a  typical 
and  severe  attack  of  spotted  fever  or  they  show  no  sign  of  infection 
whatever  and  in  the  latter  instance  it  has  been  the  custom  to  use 
them  as  duplicates  in  the  course  of  routine  passage.  Occasionally, 
however,  such  animals  have  been  found  to  be  resistant  to  infection. 
For  example,  guinea-pig  1064,  which  in  a  particular  experiment 
had  been  injected  with  o.oi  c.c.  of  infected  serum,  showed  no 
disturbance  other  than  one  day  of  distinct  fever,  the  following 
being  the  record  of  the  temperature:    103,  8,  102.8,  102.4,  102.8, 

102.6,  104.8,  102.6,  103.2,  102.8,  103.2.  Nine  days  after  the 
first  injection,  it  received  intraperitoneally  i .  25  c.c.  of  virus, 
diluted  with  salt  solution,  which  had  been  kept  in  the  ice-chest 
for  five  days.  Although  a  control  developed  the  disease,  the 
animal  was  undisturbed  by  the  inoculation,  exhibiting  normal 
temperature.  A  second  immunity  test  was  given  15  days  later 
with  the  same  result. 

One  might  be  inclined  to  explain  such  occurrences  by  the 
assumption  that  occasional  guinea-pigs  are  immune  to  the  disease 
naturally.  Two  conditions,  especially,  argue  against  the  correct- 
ness of  this  assumption:  First,  we  have  found  no  guinea-pigs  which 
are  immune  to  the  infection  when  inoculated  with  a  moderate 


350  Contributions  to  Medical  Science 

quantity  of  the  virus  taken  from  an  animal  early  in  the  disease, 
the  minimimi  quantity  necessary  for  infection  being  in  the  neigh- 
borhood of  0.02  or  0.03  c.c.  Second,  it  is  not  difficult  to  repeat 
the  result  just  quoted  by  injecting  suitable  quantities  of  virus 
and  immune  serum,  the  existence  of  active  immunity  being  proved 
by  an  immunity  test  which  is  given  after  the  passive  immunity 
estabHshed  by  the  injection  of  the  immune  serum  has  disappeared. 
This  will  be  referred  to  again  under  the  subject  of  "protective 
inoculation." 

The  active  immunity  is  probably  of  long  duration.  Animals 
have  been  found  to  resist  infection  with  excessive  doses  of  virus 
more  than  a  year  after  they  suffered  from  the  disease.  The 
occurrence  of  hereditary  immunity,  to  be  referred  to  below,  also 
indicates  the  profound  change  which  the  disease  produced  in  the 
guinea-pig  as  well  as  the  permanent  character  of  the  immunity. 

There  can  be  Httle  doubt  that  the  cause  of  the  active  immunity 
lies  in  the  anti-infectious  properties  which  may  be  demonstrated 
in  the  blood  and  serum  of  animals  which  recover,  and  perhaps 
also  in  the  acquired  power  of  the  tissues  of  the  immune  animal  to 
produce  additional  antibodies  readily  when  fresh  virus  is  introduced 
at  a  subsequent  time.  The  properties  of  the  serum  are  considered 
below  under  "Passive  Immunity."  At  present  we  have  no  means 
of  deciding  whether  the  antibodies  are  antitoxic,  germicidal,  or 
opsonic.     Theoretically  all  three  may  be  represented. 

HEREDITARY   IMMUNITY. 

The  offspring  of  a  female  guinea-pig  that  has  recovered  from 
spotted  fever  are  endowed  with  a  strong  and  protracted  immunity. 
This  has  been  demonstrated  many  times  and  the  resistance  is  trans- 
mitted regardless  of  the  degree  of  infection  of  the  female  parent, 
a  fact  which  is  shown  in  the  case  of  certain  "vaccinated"  female 
parents  which  exhibited  a  minimum  febrile  reaction.  The  influence 
of  the  immune  male  in  the  transmission  of  the  immunity  has  not 
been  investigated.     Illustrative  experiments  will  be  described. 

The  female  guinea-pig  663  was  infected  by  male  tick  No.  5  on 
June  30,  1907.  The  following  was  the  course  of  the  temperature: 
101.6,    102.4,    102.2,    103.7,    104,    105.4,    105.4,    105.2,    104.9, 


Immunity  in  Spotted  Fever  351 

104.6,  104.7,  103-8,  102.7;  recovery.  She  gave  birth  to  one 
young  on  October  14.  On  November  27,  when  the  young  animal 
was  about  six  weeks  old,  it  was  injected  intraperitoneally  with 
I  c.c.  of  third-day  virus  (Bradley  strain).  It  had  been  with  the 
parent  continuously  until  injected.  The  following  daily  tempera- 
tures were  recorded:   103,  103.6,  103.9,  I03-2,  103,  102.5,  103.4, 

103. 1,  103,  103.8,  103,  103.8,  102.6,  103. 1,  102.7.*  A  control 
guinea-pig  of  the  same  age,  from  a  normal  female,  inoculated 
with  the  same  dose  of  virus,  showed  the  following  daily  tempera- 
tures:   103,  102.8,  102.8,  105.6,  107,  106.3,  106,  104.4,  104.5, 

103.2,  100.2;  death.  The  anatomical  changes  at  autopsy  were 
typical  of  spotted  fever. 

This  experiment  is  important  as  showing  that  a  female  may 
transmit  the  immunity  to  her  young  although  her  infection  ante- 
dated the  period  of  her  pregnancy  by  several  months;  that  is,  the 
cells  of  the  embryo  were  not  stimulated  to  form  protective  sub- 
stances by  the  presence  of  virus;  only  the  germ  cell  could  have  been 
subjected  to  such  an  influence.^ 

The  following  "exchange  experiment"  indicates  that  the 
inherited  immunity  does  not  depend  entirely  on  the  milk  which 
the  young  derive  from  the  immune  parent. 

Immune  young  which  sucked  immune  parent. — Guinea-pig 
938,  30  days  old.  Sucked  for  14  days,  was  then  removed,  and 
16  days  later  was  inoculated.  Had  no  distinct  rise  in  temperature. 
Guinea-pig  939,  34  days  old.  Remained  with  parent  until  inocu- 
lated.    No  distinct  fever. 

Immune  young  which  sucked  normal  parent. — Guinea-pig  935, 

29  days  old.  Remained  with  normal  parent  until  inoculated. 
No  distinct  fever  followed.  Guinea-pig  936,  34  days  old.  Was 
with  normal  parent  for  26  days.     No  fever  followed  inoculation. 

Normal  young  which  sucked  immune  parent. — Guinea-pig  937, 

30  days  old.  Course  of  temperature  following  inoculation:  102 .8, 
102.4,  104.2,  105.3,  105.5,  105.6;  death.  Autopsy:  typical 
of  spotted  fever. 

'  In  oiir  experience  the  young  guinea-pig  frequently  has  a  higher  average  temperature  than  the  adult 
and  it  is  subject  to  greater  fluctuations. 

'  Spotted  fever  in  the  guinea-pig  is  strictly  an  acute  infection.  The  blood  of  the  animal  which 
recovers  is  never  infective  for  other  animals,  but  on  the  contrary  is  protective,  as  stated  later. 


352  Contributions  to  Medical  Science 

Normal  young  -which  sucked  normal  parent. — Guinea-pig  940,  29 
days  old.  Daily  temperatures  following  inoculation:  102.4, 
102.5,  103-8,  104.5,  105,  105. 1,  98.7;  death.  Autopsy:  typical 
of  spotted  fever. 

The  duration  of  the  inherited  immunity  has  not  been  definitely 
determined.  In  one  instance  it  was  present  two  and  one-half 
months  and  in  another  three  months  after  birth. 

We  have  not  yet  studied  the  character  of  the  inherited  immunity. 
In  the  event  that  the  infection  of  the  parent  occurred  before  her 
impregnation,  there  is  reason  to  think  that  the  immunity  of  the 
young  is  passive  in  character.  If  infection  occurs  during  preg- 
nancy, there  may  be  opportunity  for  the  establishment  of  active 
immunity  on  the  part  of  the  embryo.  However,  infection  occur- 
ring at  this  time  usually  results  in  abortion.  In  the  event  that 
the  condition  is  one  of  passive  immunity,  it  differs  from  the  passive 
immunity  established  by  the  injection  of  immune  serum  by  its 
greater  duration.  The  immunity  conferred  by  the  injection  of 
I  c.c.  of  immune  blood  into  a  guinea-pig  has  disappeared  largely 
after  the  lapse  of  30  days.  It  is  to  be  noted,  however,  that  the 
oflfspring  of  an  immune  female  may  contain  in  their  body  fluids 
a  much  larger  quantity  of  protective  substances  than  is  introduced 
in  I  c.c.  of  immune  blood,  and  that  the  elimination  of  the  larger 
quantity  may  require  a  much  longer  time.  This  subject  will  be 
investigated  further. 

passive  immunity. 

The  whole  blood,  defibrinated  blood,  and  serum  of  animals  that 
have  recovered  from  spotted  fever  possess  strong  protective  powers 
when  injected  with  virus  into  healthy  guinea-pigs.  For  the  sake 
of  convenience  immune  defibrinated  blood,  rather  than  serum,  has 
been  used  for  most  of  the  experiments,  although  it  has  been  deter- 
mined that  the  protective  powers  lie  in  the  serum.  When  the 
term  "blood"  is  used,  it  refers  to  defibrinated  blood. 

For  some  of  the  experiments  on  passive  immunity  the  blood  of 
animals  which  have  recovered  and  which  have  been  subjected  to 
no  further  treatment  has  been  used;  in  other  cases  so-called 
"hyperimmune"   blood  was   employed.      This    is    the    blood   of 


Immunity  in  Spotted  Fever  353 

guinea-pigs  that  after  recovery  received  a  series  of  injections 
of  infected  blood  with  the  hope  of  increasing  the  quantity  of  pro- 
tective substances.  We  doubt,  however,  whether  such  a  practice 
is  of  much  service  in  producing  an  increase  in  the  amount  of  anti- 
bodies. In  two  instances  the  blood  of  guinea-pigs  which  were 
treated  in  this  way  showed  an  actual  decrease  in  their  protective 
power  which  fell  below  that  of  other  immune  animals  that  had  not 
received  fresh  injections  of  virus.  Both  of  these  animals  were 
bled  repeatedly  during  the  immunizing  process,  however,  and  the 
decrease  may  have  been  due  to  the  dilution  accompanying  the 
restitution  of  the  blood.  As  a  rule,  the  blood  of  guinea-pigs  that 
have  recovered  recently  has  shown  as  great  protective  powers 
as  that  of  "hyperimmune"  animals.  It  is  not  unlikely  that  the 
concentration  of  antibodies  in  the  blood  of  the  immune  animal 
decreases  with  time.  There  is  a  possible  source  of  error  in  deter- 
mining this  point,  however,  in  that  a  number  of  successive  bleed- 
ings of  the  same  guinea-pig  brings  about  a  dilution  of  the  protective 
substances  in  his  body  simulating  a  decrease  by  elimination  or 
destruction  within  the  body.  Experiments  bearing  on  this  point 
have  not  been  brought  to  completion. 

The  blood  of  guinea-pigs  that  have  recovered  from  the  disease 
recently  protects  in  doses  of  from  o.i  to  0.3  or  0.4  c.c.  against 
infection  with  i  c.c.  of  third-day  virus,  a  quantity  which  represents 
at  least  from  30  to  60,  and  in  some  instances  100,  minimum  patho- 
genic doses.  This  result  is  obtained  when  the  immune  blood  and 
virus  are  mixed  before  injection,  the  inoculations  being  intraperi- 
toneal. Preliminary  experiments  indicate  that  the  protection  is 
not  so  pronounced  when  the  two  are  injected  into  different  parts 
of  the  body  at  the  same  time,  although  the  difference  is  not  great. 

The  following  experiment  gives  an  approximate  idea  of  the 
duration  of  the  passive  immunity  when  i  c.c.  of  immune  blood  is 
injected  subcutaneously  (Table  i).  The  immune  blood  in  this 
instance  was  a  mixture,  obtained  from  three  immune  guinea-pigs 
(862,  878,  778),  two  of  which  had  recovered  from  spotted  fever 
one  month  previously  and  the  third  about  three  months  previously. 
One-tenth  cubic  centimeter  protected  against  i  c.c.  of  third-day 
virus  in  controls,  the  mixtures  being  injected  intraperitoneally. 


354 


Contributions  to  Medical  Science 


The   experiment   animals   received   the   virus   subcutaneously   at 
different  periods  following  the  injection  of  the  immune  blood. 

The  relation  of  the  duration  of  the  passive  immunity  to  the 
quantity  of  immune  serum  injected  has  not  been  determined. 


TABLE  I. 
Duration  of  Passive  Immunity. 


Guinea-Pig 

Interval  between  Injec- 
tion of  Serum  and  Virus 

Quantity  of 
Virus  Injected 

Result 

20  days 

33 

38 

45 

49 

55 

I.O  c.c. 

I.O 
l.O 
I.O 

o-S 
0.03 

Slight  fever  for  5  days 
Moderate  fever  for  s  days 
Severe  fever  for  9  days  ' 

I002 

period  of  one  week 

The  converse  of  this  experiment  was  performed  also;  a  number 
of  guinea-pigs  were  inoculated  subcutaneously  with  i  c.c.  of  virus 
each,  and  the  subsequent  period  determined  at  which  i  c.c.  of 
immune  blood  would  prevent  the  development  of  spotted  fever 
(Table  2).  In  this  case  the  immune  bloods  were  drawn  imme- 
diately before  injection  and  they  came  from  different  immune 
animals;  hence  they  probably  were  not  of  uniform  value,  though 
certainly  not  differing  greatly. 

TABLE  2. 
Prophylactic  Effect  of  Immune  Blood  when  lN.fEcrED  Subsequent  to  Virus. 


Guinea-Pig 

Interval  between  Injec- 
tion of  Virus  and 
Immune  Blood 

Result 

851 

1  day 

2  days 

3 
4 

852 

other  causes 

8s3 

the  same  day 

8s4 

three  days  of  low  fever  (about  104) 

The  experiment  shows  protection  against  30  to  60  pathogenic 
doses  at  the  least,  when  i  c.c.  of  immune  blood  is  given  three  days 
after  the  injection  of  the  virus.  From  this  time  the  injections  of 
immune  blood  could  be  considered  to  have  only  a  curative  effect, 
and  this  will  be  mentioned  later. 


Immunity  in  Spotted  Fever  355 

The  results  of  these  experiments  on  protection  by  means  of 
immune  blood  or  serum,  of  which  we  have  abundant  confirmation, 
would  seem  to  have  an  important  practical  bearing.  There  is 
good  reason  to  believe,  on  the  basis  of  the  results  obtained,  that 
serum  prophylaxis  of  man  is  feasible,  provided  sufficient  quantities 
of  a  serum  of  reasonable  strength  can  be  prepared.  Manifestly 
it  would  operate  successfully  only  in  case  the  inhabitants  of  infected 
districts  who  are  bitten  by  ticks  would  report  for  prophylactic 
injections  within  two  or  three  days  after  receiving  the  bite.  If 
the  results  obtained  with  the  guinea-pig  apply  also  to  man,  the 
serum  when  given  in  sufficient  quantity  would  ward  off  the  danger 
pertaining  to  the  recent  exposure,  but  probably  could  not  be 
considered  protective  for  a  period  longer  than  three  or  four  weeks. 
A  subsequent  exposure  would  require  another  injection  of  serum. 

We  have  been  able  to  infect  the  horse  with  spotted  fever  recently 
and  are  now  engaged  in  a  study  of  the  protective  power  of  the 
serum  obtained  after  the  recovery  of  the  animal.  Preliminary 
experiments  indicate  that  its  value  differs  Httle  from  that  of  the 
guinea-pig,  hence  there  is  reason  to  believe  that  a  protective 
serum  in  desirable  quantities  will  be  available. 

AN   EXPERIMENT   IN   SERUM   THERAPY. 

We  have  made  preliminary  observations  on  the  curative  value 
of  the  immune  blood  from  the  guinea-pig  and  shall  describe  an 
experiment  which  seems  to  indicate  that  its  therapeutic  power 
is  not  of  high  degree  (Table  3). 

This  is  a  continuation  of  the  passive  immunity  experiment 
described  on  p.  354,  in  which  equal  quantities  (i  c.c.)  of  virus  were 
inoculated  into  a  number  of  guinea-pigs  and  the  effect  of  the 
immune  blood  noted  when  given  at  subsequent  periods.  Inasmuch 
as  the  remaining  animals  began  to  show  fever  on  the  fourth  day 
after  inoculation,  it  became  thereafter  an  experiment  to  determine 
the  curative  value  of  the  immune  blood. 

It  had  been  determined  in  other  experiments  that  the  normal 
blood  of  the  guinea-pig  has  no  protective  power  against  spotted 
fever.  Thus  guinea-pig  884  received  intraperitoneally  a  mixture 
of  I  c.c.  of  normal  blood  and  0.3  c.c.  of  third-day  virus  and  died 


356 


Contributions  to  Medical  Science 


of  spotted  fever  after  nine  days.  Also  guinea-pig  886  received 
I  c.c.  of  normal  blood  and  0.05  c.c.  of  virus  and  exhibited  the 
course  characteristic  of  spotted  fever,  including  hemorrhage  into, 
and  sloughing  of,  the  scrotum.  It  resisted  an  immunity  test 
given  two  months  after  recovery. 

TABLE  3. 
The  Curative  Power  of  Immuke  Blood. 
Virus  injected  December  4,  1907,  subcutaneously. 
Injections  of  immune  blood  subcutaneously  on  dates  indicated. 
Guinea-pigs  designated  by  numbers,  855,  etc. 


Dec. 


Temp. 

Immune 

Temp. 

Immune 

Temp. 

Immune 

Temp. 

8SS 

Blood 

856 

Blood 

8S7 

Blood 

858 

103.8 

103.4 

104. 

103 -9 

103.4 

104. 

103.7 

103.4 

103.5 

104.2 

104.9 

104.8 

103.6 

103. 

103.7 

103.1 

105.6 

2  c.c. 

105.8 

sec. 

105. 

1  c.c. 

105.8 

105.5 

los- 

105.7 

106. 

105.7 

105. 

106.3 

105.7 

104.  s 

103.4 

104.8 

104.6 

104.2 

103.9 

104.2 
104. 

104.2 
104.8 

102.2 

102.5 

102.9 

103.9 

102.3 

ioa.3 

103.1 

103.6 

102.  2 

102. 

102. 5 

103.  S 

102.6 

102.6 

103.7 

103.3 

103. 

102.3 

Recovery 

Recovery 

Recovery 

Recovery 

Immune 
Blood 


5 

6 

7 

8 

9 

10 

II 

12 

13 

14 

IS 

16 

17 

18 

19 


Dec. 

Temp. 
859 

Immune 
Blood 

Temp. 
860 

Immune 
Blood 

Controls* 

103.3 

104. 

104.4 

103.7 

105.6 

106.4 

106.8 

106. 

105.8 

105.2 

103.6 

103.2 

102.7 

102.8 

Recovery 

2  c.c. 
2 
2 
2 

103.4 

103.7 

104.2 

103.6 

104.3 

106. 

106.2 

105.2 

10S.3 

105.8 

103.9 

104. 

103.1 

102.7 

103.3 

Recovery 

3  c.c. 
3 
3 
3 

103.9 

102.7 

104. 

103.  S 

105.2 

106.3 

106. 

105.4 

105.6 

103.2 
103.4 
103. 
102.7 

Recovery 

103.6 

6:::::::::::::::::::::::::: 

104. 

104. 

8': :::::::::::::::::: 

103.7 

106.4 

106.2 

105.7 

105.1 

105.8 

103. 

16"  ;■ ::::::::::::::::::::: 

103.  S 

103. 

18        

103. 1 

Recovery 

*  Inoculated  with  i  c.c.  of  virus  alone. 


Inasmuch  as  both  controls  in  this  experiment  recovered,  the 
influence  of  the  immune  blood  can  be  determined  only  by  its 
effect  on  the  duration  and  severity  of  the  fever,  the  normal  duration 
of  which  in  this  instance  was  approximately  six  days.     With  this 


Immunity  in  Spotted  Fever  357 

as  a  basis  of  comparison,  it  is  seen  that  2  c.c.  of  immune  blood 
given  on  the  first  day  of  fever  exercised  but  slight  if  any  influence 
on  the  course.  Five  cubic  centimeters  given  on  the  first  day 
quite  positively  shortened  the  course,  since  there  were  only  three 
days  of  severe  fever.  One  cubic  centimeter  given  daily  had  no 
pronounced  influence,  although  the  temperature  for  the  last  three 
days  was  lower  than  in  the  controls.  Two  cubic  centimeters 
given  daily  beginning  with  the  second  day  of  fever  may  have 
been  a  factor  in  the  lower  temperature  during  the  last  three  days. 
The  same  amount  given  daily,  beginning  on  the  third  day  of  fever, 
had  no  effect,  and  a  similar  result  is  noted  when  the  amount  was 
increased  to  3  c.c.  daily. 

The  experiment  cited  seems  to  show  quite  positively  that  the 
immune  blood  exercises  little  influence  on  the  disease  unless  it 
can  be  given  early  and  in  large  quantities.  An  important  practical 
difficulty  arises  here,  for  the  disease  in  man  is  rarely  recognized 
until  the  eruption  appears,  which  may  be  at  any  time  from  the 
second  to  the  fifth  day  of  fever.  Physicians  who  are  experienced 
in  the  diagnosis  of  the  infection  often  make  a  probable  diagnosis 
of  spotted  fever  on  the  first  or  second  day  of  sickness,  in  the  spring 
of  the  year,  in  the  case  of  patients  that  reside  in  infected  districts. 
The  probable  diagnosis  should  be  facilitated  if  a  history  of  tick 
bite  within  one  to  two  weeks  preceding  the  onset  of  symptoms  is 
given.  In  such  cases,  a  large  quantity  of  the  serum  given  imme- 
diately may  exercise  a  modifying  influence  on  the  course  of  the 
infection;  in  the  event  that  the  probable  diagnosis  was  incorrect, 
there  would  be  no  Hkelihood  of  harm  as  a  consequence  of  the 
injection  of  the  serum. 

The  curative  value  of  the  immune  serum  from  the  horse  is  being 
studied  carefully;  inasmuch,  however,  as  the  preliminary  tests 
have  shown  that  it  possesses  about  the  same  power  as  the  blood 
of  immune  guinea-pigs,  it  is  not  anticipated  that  the  results  will 
be  very  different. 

It  is  not  probable  that  a  serum  of  greater  power  than  that 
afforded  by  the  animal  which  has  recovered  from  spotted  fever 
will  be  obtained  until  the  micro-organism  can  be  grown  in  desirable 
quantities  in  artificial  cultures.    During  the  course  of  the  infec- 


358  Contributions  to  Medical  Science 

tion  there  is  already  in  the  animal's  body  such  a  quantity  of  the 
organism  that  0.02  or  0.03  c.c.  of  his  blood  is  infective,  and  at 
present  there  is  no  means  of  increasing  this  amount  for  subsequent 
injections. 

protective  inoculations. 

For  more  than  a  year  occasional  attempts  have  been  made  to 
attenuate  the  virus  of  spotted  fever  by  desiccation,  by  the  addition 
of  glycerin,  and  by  means  of  heat  so  that  it  would  be  suitable  for 
vaccination.  Desiccation  for  this  purpose  has  proved  to  be  utterly 
unreliable  and  has  been  abandoned.  The  experiments  will  not 
be  described  further  than  to  say  that  the  principle  followed  was 
to  begin  the  injections  with  comparatively  large  quantities  of 
virus  that  had  been  dried  in  a  vacuum  over  sulphuric  acid  for  a 
longer  period  than  was  required  to  kill  all  the  organisms,  and  for 
succeeding  injections  to  use  smaller  quantities  of  virus  which  had 
been  dried  for  shorter  periods,  passing  finally  to  minute  amounts 
of  fresh  virus.  Infection  eventually  resulted  in  practically  all  the 
animals,  and  although  it  may  in  the  end  be  possible  to  produce 
immunity  in  this  way,  the  method  is  so  tedious,  long,  and  uncertain, 
that  it  appears  to  have  only  theoretical  interest. 

The  value  of  glycerin  and  low  degrees  of  heat  as  attenuating 
agents  is  still  being  studied. 

No  systematic  attempt  has  been  made  to  determine  the  vac- 
cinating properties  of  minute  doses  of  the  virus.  As  a  rule 
a  minute  quantity  either  produces  frank  infection  or  causes  no 
disturbance  whatever,  and  in  the  latter  case  the  animals  are,  in 
nearly  all  instances,  susceptible  to  reinoculation.  Occasional 
exceptions  have  been  encountered,  however.  Such  an  animal 
(guinea-pig  1064)  was  referred  to  under  the  discussion  of  active 
immunity  (p.  349).  In  this  instance  marked  active  immunity 
developed,  although  the  animal  had  but  one  day  of  fever.  On 
account  of  the  uncertainty  as  to  what  the  virus  will  do  when 
injected  in  quantities  which  approximate  the  minimum  patho- 
genic dose,  it  is  manifest  that  minute  doses  cannot  be  utilized  for 
practical  vaccination. 

On    account   of   the   protective   power   which   immune   blood 


Immunity  in  Spotted  Fever  359 

possesses,  the  possibility  of  mixed  immunization  or  ''sero-vac- 
cination"  seemed  promising  and  we  have  devoted  some  time  to 
the  study  of  this  subject. 

At  the  beginning  of  the  work  three  plans  of  inoculation  were 
adopted:  First,  the  immunizing  effect  of  a  single  mixture  of 
immune  blood  and  virus.  Second,  the  effect  of  repeated  injec- 
tions given  at  short  intervals,  the  ratio  of  the  quantity  of  the  im- 
mune blood  to  the  virus  being  gradually  decreased  until  pure 
virus  was  injected.  Third,  the  same  as  the  second  plan,  except 
that  longer  intervals  were  allowed  to  intervene  between  the  injec- 
tions. The  ultimate  object  in  all  three  methods  was  to  establish 
active  immunity  without  causing  severe  infection.  This  was 
found  to  be  readily  possible  with  regard  to  the  second  and  third 
methods,  although  the  development  of  immunity  in  most  instances 
was  characterized  by  a  mild  and  brief  febrile  reaction  some  time 
during  the  course  of  the  injections.  These  methods,  however,  are 
protracted  and  tedious  and  are  more  of  theoretical  than  of  practical 
interest,  particularly  since  a  high  degree  of  immunity  can  also 
be  established  by  a  single  injection  of  virus  and  immune  blood 
when  the  two  are  used  in  proper  proportions. 

We  shall  therefore  limit  our  description  to  certain  experiments 
in  which  immunity  was  established  in  one  instance  by  two  injec- 
tions (Table  4),  and  in  another  by  one  injection. 

Following  the  second  injection  all  the  animals  showed  a  certain 
amount  of  febrile  reaction,  although  in  the  first  three  the  rise  in 
temperature  was  no  greater  than  that  often  encountered  from  other 
causes,  such  as  minor  injuries  to  the  colon  by  the  thermometer. 

It  was  attempted  to  learn  the  degree  of  immunity  which  had 
been  established  in  789,  which  showed  a  minimum  reaction,  and 
in  791,  which  showed  a  more  pronounced  though  mild  reaction, 
by  testing  the  protective  power  of  their  defibrinated  bloods,  in 
two  consecutive  experiments  (Tables  5  and  6) . 

The  first  test,  shown  in  Table  5,  is  not  very  satisfactory,  particu- 
larly regarding  the  animals  injected  with  the  blood  of  guinea-pig 
791.  Guinea-pig  960  which  received  the  largest  dose  of  this  blood 
(i  c.c.)  ran  a  distinct  course  of  fever,  the  cause  of  which  may  not 
be  open  to  determination.    When  sufficient  time  has  elapsed  for 


360 


Contributions  to  Medical  Science 

TABLE  4. 
The  Immunizing  Effect  of  Two  Injections. 


First  injection,  consisting  of  virus  mixed  with  different  quantities  of  immune  blood.    Injections 
intrapertJoneally.    Immune  blood  from  two  animals  (765,  766)  that  had  recovered  spontaneously. 


No.  of  Experiment  Animal 

Virus 

Immune  Blood 

Result 

1  c.c. 

I 
I 
1 

I.S  c.c. 
1.0 
0-7 
0-3 

No  fever 

"      " 

"      " 

"      " 

Second  injection,  consisting  of  i  c.c.  of  virus,  given  25  days  after  the  first. 


Date 

Guinea-Pig 
789 

Guinea-Pig" 
790 

Guinea-Pig 
791 

Guinea-Pig 
792 

Dec.  2 

102.0 
102.3 
102.4 
102.1 
101.8 
103.6 
102.8 
102.8 

103.8 
102.7 

102.2 

'°^/; 
102.0 

102.6 

102.6 

103.4 

103.8 

103.2 

103.4 

103.7 

102.7 

102.2 

103. 

102.7 

102.3 

102.4 

103. 

103.4 

104.2 

103.9 

104. 

102.2 

102.4 

103. 

102.8 

103. 

i.::.'.'.:'.'.'.'.'.'.'. 

104.7 

105.5 

8 

104. 

104.8 

103.3 

94.4 

Death 

TABLE  5. 

The  Protective  Power  of  the  Bloods  of  the  Vaccinated  Guinea-Pigs  789  and  791. 

Test  1,  performed  25  days  after  the  second  injection. 
Virus,  1  c.c.-1-varying  quantities  of  immune  blood. 


Date 

Immune  Blood  from 
Guinea-Pig  789 

Immune  Blood  from 
Guinea-Pig  791 

Controls,  Re- 
ceiving I  c.c.  OF 
Virus  EAcnf 

I  c.c. 

No.  957 

0.7  c.c. 
No.  958 

0.3  c.c. 
No.  959 

1  c.c. 
No.  960 

0.7  c.c. 
No.  961 

0.3  c.c. 
No.  962 

No.  965 

No.  966 

Dec.  27 

29 

30 

T       31 

Jan.     I 

2 

3 

4 

s 

6 

7 

8 

9 

10 

II 

102.8 
102.9 
102.6 
102.6 
102.4 

102.4 

103.2 
102.8 

102.6 

102.4 
101.6 
103.2 

Recovery 

102.8 
103.1 
103. 
103.6 
103.  S 

103. 

104.4 
104.3 

104. 
103.4 
103.5 
103. 1 
103. 5 
Recovery 

103.0 
103.2 
104.3 
105.2 
103.9 

104.8 

104. 
102.8 

103.6 
104.3 
103.2 
104.3 
104. 
Recovery* 

103.0 
103.  S 
103.8 
104. 
103.5 

104.7 

104.3 
104.5 

104. 
104.7 
104.8 
103.9 
102.8 
Recovery 

102.8 

103.3 

104. 

104. 

103.4 

103.2 

103.2 
102.5 

102.3 
102.4 
102.3 
103. 

Recovery 

102.7 

102.8 

102.8 

103. 

103.1 

102.7 

102!  8 
102.4 

102.2 
102.4 
102.6 
103.4 

Recovery 

102.4 

102.6 

102.4 

103. 

104.4 

105.2 

105.4 

KiUed 

103.6 

103.8 

102.8 

103.2 

104. 

105.4 

•  Guinea-pig  959  died  two  weeks  later,  showing  pneumonia  and  cheesy  abscesses, 
t  Guinea-pigs  965  and  966  were  "passage"  animals  and  they  were  killed  on  January  2  in  order  to 
perpetuate  the  strain  in  other  guinea-pigs. 


Immunity  in  Spotted  Fever 


361 


TABLE  6. 
The  Protective  Power  of  the  Bloods  of  the  Vaccinated  Guinea-Pigs  789  and  791. 
Test  2,  performed  29  days  later  than  the  first  test,  and  54  days  after  the  second  immunizing  injec- 
tion was  given. 

Virus,  I  c.c. -(-varying  amounts  of  the  immune  bloods. 


Date 

luvuNE  Blood  from 
GniNEA-PiG  789 

luuxTNE  Blood  from 
GuiNEA-PiG  791 

Controls  = 
Virus  Alone 

0.6  c.c. 
No.  1074 

0.4  c.c. 
No.  1073 

0.2  c.c. 

No.  1072 

0.5  c.c. 
No.  1077 

0.3  c.c. 
No.  1076 

0.1  c.c. 
No.  1075 

o.oi  c.c. 
No.  1078  • 

0.05  c.c. 
No.  1079 

Jan.  25 

26 

'°3„ 
102.8 

103. 

102.6   .g 

103.4  00 
103.4    c 
104.      « 
104.4   « 
103.6  ^ 
105.2    ° 
104.4    0 
104.4  '^ 
102.4    ^ 

102.2      Q 
102.4 

Recovery 

103.8 
103.2 
103.2 
103.2    ?, 
102.4  ^ 
103.2    0. 

104.4    c 

104.2  « 

105.3  ^ 
104.8  .„ 

^°5-      ° 
104.      0 

103.4  '^ 
103.6    5 

102.8     Q 
102. 

Recovery 

i«3-6 

102.6 

102.6  g, 
102.     « 
105.     0, 

104 . 8    ^- 
105.6    « 
104.4    " 
104.     i^ 
104.4    ° 
104.6    § 
104.4  "5 
103.8   2 
102.8  (5 
102.8 

Recovery 

102.8 

102.6 
102.6 
102.8 
102.8 
102.8    ^ 

103.6  ^ 

102.7  ^ 

102.8  0 

102.  "= 

103.  0 
102.8     J 
102.8    ■= 
102.2    -^ 
102.3 

Recovery 

102.6 
101.8 
102.2 

102.8  5. 
102.6  .g 
103 . 2  ^ 

102.8  « 
103.8  t 
104.    .*^ 
104.6  ° 
104.      s 

103.4  "5 
102.8  2 

102.6 

Recovery 

103.2 
102.      g. 
102.     ^ 
103.4   ■.» 
102.4   g 

'O 

103.2    . 
105.     " 

104.8   Z 

103.8  :n 
104.    ° 
104.    § 

102.8  'S 

X02.    2 
102.   p 
102.3 

Recovery 

102.8 
102.4 
102.2 
102.4 
100.8 
102. 
102.4 
103.2 
102.4 
102.4 
103.2 
103.4 
103.6 
102.2 
102.4 
102.4 
Recovery 

103.2 

102.2 

a8 

104.6 

105.2 

Feb.  I 

105.8 

104. 

99.8 
Death  t 

6 

8 

•  Guinea-pig  1078  became  infected  when  an  immunity  test  was  given  on  February  28,  hence  in  this 
experiment  the  minimum  pathogenic  dose  was  greater  than  o.oi  c.c.  of  virus,  but  equal  to  or  less  than 
0.05  c.c.  as  shown  by  the  fatal  course  in  guinea-pig  1079. 

t  The  genitalia  were  hemorrhagic,  and  the  spleen  was  greatly  enlarged. 


the  passive  immunity,  which  was  conferred  by  the  immune  blood, 
to  be  eliminated,  the  protective  power  of  its  blood  will  be  ascer- 
tained, and  if  this  proves  to  be  of  sufficient  strength  it  will  have  to 
be  concluded  that  the  animal  suffered  from  a  moderately  severe 
attack  of  spotted  fever,  in  spite  of  the  large  dose  of  protective 
blood  which  it  received,  and  in  spite  of  the  fact  that  one-third  of 
this  dose  protected  guinea-pig  962. 

Having  in  mind  the  animals  which  received  0.7  and  0.3  c.c.  of 
the  two  immune  bloods,  the  impression  is  given  that  the  blood  of 
791  was  somewhat  more  protective  than  that  of  789. 

The  difference  in  the  protective  power  of  the  two  bloods  is 
manifest  in  the  second  test.  Thus,  0.5  c.c.  of  the  blood  of  791 
prevented  fever  entirely,  whereas  0.6  c.c.  of  789  did  not  prevent 
the  development  of  a  distinct  and  rather  high  course  of  fever; 
0.3  c.c.  of  the  former  permitted  moderate  fever  for  five  days, 
while  0.4  c.c.  of  the  latter  allowed  a  marked  febrile  course  of  nine 
days;  and  the  conditions  are  similar  in  regard  to  the  third  animal 


\62 


Contributions  to  Medical  Science 


of  each  group  (1075  and  1072).  One  probably  could  not  come 
nearer  to  a  comparison  than  to  say  that  0.6  c.c.  of  the  blood  of 
guinea-pig  789  was  equal  to  o.i  c.c.  of  that  from  791,  since  the 
severity  and  duration  of  the  fever  of  the  corresponding  animals  were 
about  the  same. 

Comparison  of  the  first  test  with  the  second  indicates  that  the 
protective  substances  underwent  a  diminution  in  quantity  in  the 
interval  of  29  days.  In  the  first  test  the  blood  of  guinea-pig  791 
in  a  dose  of  0.3  c.c.  prevented  fever  entirely,  with  the  possible 
exception  of  two  days  when  the  temperature  was  103  and  103 .  i 
respectively.  In  the  second  test  the  same  quantity  of  blood 
permitted  a  distinct  though  moderate  fever  for  five  days.  Simi- 
larly, 0.3  c.c.  of  the  blood  of  guinea-pig  789  in  the  first  test  afforded 
greater  protection  than  0.6  c.c.  in  the  second  test.  The  conclu- 
sion that  such  a  diminution  does  occur  has  been  borne  out  in  other 
experiments. 

Such  comparative  experiments,  performed  at  different  times, 
are  subject  to  possible  error  in  that  the  minimum  pathogenic  dose 
of  two  different  lots  of  virus  may  not  be  identical.  In  our  experi- 
ence, however,  this  variation  has  been  slight  and  certainly  would 
not  cause  greater  error  than  would  result  from  slight  diff'erences 
in  the  susceptibility  of  different  animals. 

An  experiment  similar  to  the  one  just  described  was  carried  on 
with  guinea-pigs  835,  836,  and  837,  only  the  essential  points  of 
which  will  be  given. 

On  November  25  all  three  were  injected  with  i  c.c.  of  virus 
to  which  had  been  added  different  quantities  of  immune  blood; 
for  835,  0.3  c.c,   836,  o.  I  c.c,  and  837,  0.05  c.c. 

Guinea-pig  835  had  no  fever  following  the  injection.  Guinea- 
pig  836  showed  the  following  course  of  temperature  on  successive 
days:  103.4,  103.7,  103,  103,  102.9,  103 -8,  103.4,  103.8,  104.4, 

104.5,  103 -3'  102.8,  103.2,  102.6.  The  fever  traceable  to  the 
virus  was  brief  and  mild  in  character.  Guinea-pig  837  suffered 
severe  infection  with  the  following  course  of  the  temperature: 
102.9,    102.8,    102.9,    103. I,    102.8,    105,    105.6,    105.8,    104.9, 

104.6,  105. I,  104.2,  103.4,  103,  103.3,  102.6,  102.6. 


Immunity  in  Spotted  Fever  363 

They  received  no  further  inoculations  of  virus,  hence  the  experi- 
ment represents  an  attempt  to  immunize  by  one  injection. 

The  first  comparative  test  of  the  protective  powers  of  the  bloods 
of  the  three  animals  was  made  on  January  7,  about  two  months 
after  the  injections  were  made.  This  was  ample  time  for  all  the 
passive  immunity  to  have  disappeared,  as  shown  by  an  experiment 
quoted  earlier  in  this  paper.  Suitable  quantities  of  the  different 
bloods  were  not  used  to  afford  intelHgent  comparison  of  their 
properties.  The  second  test,  performed  February  5,  showed  that 
2  c.c.  of  the  blood  of  835  had  no  protective  effect,  whereas  0.8 
c.c.  from  836,  and  0.5  c.c.  from  837  showed  moderate  protection. 
In  the  meantime  an  immunity  test  was  given  to  835,  which  resulted 
in  its  infection  with  spotted  fever,  hence  it  may  be  concluded 
that  the  initial  injection,  the  immunizing  ( ?)  injection,  which 
was  followed  by  no  fever,  had  produced  little  or  no  immunity  in 
this  animal. 

A  third  test  of  the  blood  of  836  and  837  showed  that  0.9  c.c. 
from  836  did  not  afford  complete  protection,  whereas  0.5  c.c.  from 
837  entirely  prevented  the  development  of  fever  with  the  dosage  of 
the  virus  used,  namely,  i  c.c.  of  third-day  virus.  This  test  was 
made  three  months  after  the  immunizing  injection  was  given. 

Both  of  the  experiments  described  indicate  that  there  is  a  parallel 
between  the  severity  of  the  reaction  following  the  immunizing 
injection  and  the  subsequent  protective  power  of  the  blood,  and 
it  is  reasonable  to  suppose  that  the  degree  of  immunity  conferred 
by  the  vaccination  corresponds  to  a  certain  extent  with  the  concen- 
tration of  the  protective  substances  which  appear  in  the  blood. 
This  does  not  mean,  however,  that  a  severe  or  even  moderate 
febrile  reaction  is  necessary  in  order  that  distinct  immunity  be 
conferred.  It  has  happened  frequently  that  a  barely  perceptible 
reaction  is  followed  by  the  development  of  pronounced  resistance. 
Two  instances  may  be  cited  in  which  the  immune  blood  was  given 
a  few  days  in  advance  of  the  virus. 

On  December  i,  1907,  guinea-pigs  844  and  845  received  each 
I  c.c.  of  immune  blood  subcutaneously.  Three  or  four  days  later, 
respectively,  each  was  given  i  c.c.  of  third-day  virus  (Eddy  strain) 


364  Contributions  to  Medical  Science 

subcutaneously.  The  temperature  of  guinea-pig  844  was  as  fol- 
lows: 102,  102.8,  103.4,  103.3,  103-2,  101.8,  103.3,  103-2,  102, 
102.7.  That  of  guinea-pig  845  was:  102.9,  103.4,  102.7,  103.3, 
101.7,  102.7,  102.9,  102.5,  102.9.  Sixty-seven  days  later  each 
received  intraperitoneally  i  c.c.  of  third-day  virus,  one  being 
given  the  Eddy  and  the  other  the  Bradley  strain.  Neither  showed 
the  sHghtest  febrile  disturbance.  Controls  developed  the  disease 
typically. 

hereditary  immunity  in  vaccinated  animals. 

Inasmuch  as  strong  immunity  may  be  produced  in  guinea-pigs 
by  the  method  of  "sero-vaccination,"  in  which  only  a  minimum 
febrile  reaction  is  necessary,  it  would  be  expected  that  the  immunity 
conferred  on  females  in  this  way  would  be  conveyed  to  their 
oJBfspring.  This  would  be  anticipated  from  the  fact  that  such 
a  transfer  occurs  in  the  case  of  females  which  have  suffered  from 
a  severe  attack  of  the  disease,  as  described  on  a  previous  page. 

This  phenomenon  was  noted  in  relation  to  guinea-pig  791, 
whose  history  has  been  given  already,  and  to  guinea-pig  794,  which 
had  been  immunized  by  a  series  of  graded  injections  in  which  the 
quantity  of  immune  serum  was  reduced  until  pure  virus  was  admin- 
istered. 

Two  young  were  born  to  guinea-pig  791  on  December  30,  1907. 
This  was  approximately  one  month  after  she  received  the  second 
injection  consisting  of  virus  alone  and  about  two  months  after 
the  mixed  injection  was  given.  One  of  the  young  (1080)  when 
25  days  old  received  0.5  c.c.  of  virus  intraperitoneally.  Its  tem- 
perature and  appearance  were  unchanged  by  the  injection,  whereas 
a  control  of  the  same  age  (1087)  which  received  the  same  dose 
died  of  spotted  fever  10  days  later.  The  remaining  animal  (1081), 
when  52  days  old,  was  given  an  intraperitoneal  injection  of  i  c.c. 
of  virus.  No  fever  resulted  and  the  guinea-pig  is  still  living;  the 
control  acquired  a  severe  infection.  The  same  result  was  obtained 
with  one  of  the  young  of  guinea-pig  794,  24  days  after  its  birth. 

The  experiments  quoted,  and  others  which  it  seems  unnecessary 
to  describe,  show  that  successful  sero- vaccination  of  the  guinea- 
pig  is  possible.     We  are  studying  this  problem  further  to  determine 


Immunity  in  Spotted  Fever  365 

the  applicability  of  the  method  to  man,  paying  particular  atten- 
tion to  methods  of  standardization  of  the  virus  and  immune  serum, 
the  durability  of  the  protective  substances  under  different  condi- 
tions of  preservation  and  the  constancy  with  which  a  desired 
reaction  can  be  obtained.  It  is  our  intention  at  the  same  time  to 
investigate  the  behavior  of  the  monkey  to  this  method  of  vac- 
cination. 

It  does  not  follow  that  one  can  pass  directly  from  the  guinea- 
pig  to  man  or  from  the  monkey  to  man  in  sero- vaccination,  using 
corresponding  proportions  of  virus  and  immune  serum.  The 
unknown  susceptibility  of  man  in  comparison  with  that  of  the 
monkey  and  guinea-pig  is  a  serious  stumbHng-block  in  this  con- 
nection. A  mixture  which  is  neutral  for  the  guinea-pig  or  which 
produces  only  a  slight  reaction  in  it  may  produce  a  severe  reaction 
in  man.  The  converse  may  also  be  true:  that  a  dosage  or  a 
proportion  of  constituents  which  would  excite  an  immunizing 
reaction  in  the  guinea-pig  or  monkey  would  be  without  effect  in 
man.  There  is  also  a  further  possibility  to  face,  namely,  that  the 
virus  as  a  result  of  cultivation  in  the  guinea-pig  may  have  under- 
gone modifications  in  its  virulence  whereby  it  may  have  become 
less  virulent  or  more  virulent  for  man. 

Only  one  method  could  possibly  be  advocated  at  the  outset: 
namely,  to  use  such  proportions  of  virus  and  immune  serum  as 
would  leave  no  question  as  to  the  safety  of  the  procedure,  assum- 
ing for  the  time  that  the  virus  has  the  greatest  possible  virulence 
for  man.  An  index  of  the  effect  of  such  injections  could  be  obtained 
by  studying  the  properties  of  the  resulting  serum.  In  the  event 
that  no  antibodies  were  formed  it  would  then  appear  justifiable  to 
decrease  the  proportion  of  immune  serum  to  that  of  virus,  again 
studying  the  effect  of  the  injection  on  the  properties  of  the  serum. 
This  process  could  be  continued  until  a  mixture  is  obtained  which 
causes  the  appearance  of  antibodies  without  exciting  a  severe 
reaction. 

SUMMARY. 

An  attack  of  spotted  fever  in  the  guinea-pig  and  monkey  pro- 
duces a  strong  active  immunity  of  long  duration.  This  immunity 
is  characterized  by  the  presence  of  protective  antibodies  in  the 


366 


Contributions  to  Medical  Science 


serum  which  may  be  demonstrated  by  injecting  mixtures  of  virus 
and  immune  serum.  The  concentration  of  the  antibodies  in  the 
blood  of  the  immune  animal  undergoes  a  decrease  in  the  course  of 
several  weeks. 

The  female  that  has  recovered  from  spotted  fever  transmits 
immunity  to  her  young.  The  young  are  immune  even  when  the 
female  acquired  her  immunity  several  months  before  impregnation. 
The  immunity  of  the  young  does  not  depend  on  the  ingestion  of 
milk  from  the  immune  mother.  The  character  of  the  inherited 
immunity  has  not  yet  been  determined,  although  it  is  presump- 
tively a  passive  immunity  that  differs  from  the  passive  immunity 
conferred  by  the  injection  of  immune  serum  by  its  longer  duration. 
The  long  duration  of  the  inherited  immunity  may  depend  on  the 
longer  time  required  for  the  elimination  of  large  quantities  of 
protective  substances. 

Passive  immunity  may  be  established  in  the  healthy  guinea- 
pig  by  the  injection  of  blood  or  serum  from  the  immune  guinea-pig. 
The  immune  defibrinated  blood  contains  antibodies  in  such  con- 
centration that  O.I  c.c.  often  protects  against  i  c.c.  of  third-day 
virus,  representing  anywhere  from  30  to  100  minimum  pathogenic 
doses.  In  other  instances  0.3  or  0.4  c.c.  of  immune  blood  are 
required  for  this  degree  of  protection.  When  i  c.c.  of  strong 
immune  blood  is  injected  subcutaneously  into  healthy  guinea-pigs 
the  passive  immunity  is  still  present  in  marked  degree  after  20 
days;  after  38  days  it  is  present  only  in  such  degree  that  a  mild 
course  of  spotted  fever  results  when  virus  is  injected;  after  45 
days  it  is  no  longer  manifest.  It  is  possible  that  passive  immunity 
would  not  last  so  long  if  the  immune  blood  were  injected  into  a 
foreign  species. 

The  guinea-pig  may  be  protected  against  spotted  fever  following 
its  inoculation  with  infected  blood,  provided  the  immune  blood 
is  administered  on  the  second  or  third  day  after  inoculation. 

The  curative  power  of  the  immune  blood  or  serum  is  low  and 
in  order  to  produce  a  distinct  effect  it  is  necessary  to  begin  its 
administration  early  in  the  disease  and  to  give  relatively  large 
quantities.  It  exerts  a  modifying  effect  on  the  severity  of  the 
infection  without  bringing  about  rapid  subsidence  of  the  symptoms. 


Immunity  in  Spotted  Fever  367 

By  the  method  of  mixed  immunization  or  sero- vaccination,  in 
which  virus  and  immune  blood  or  serum  are  mixed  in  suitable 
proportions,  it  is  possible  to  immunize  the  guinea-pig  by  one  or 
several  injections,  with  the  result  that  he  is  thereafter  immune 
to  infection.  The  blood  of  animals  immunized  in  this  way  con- 
tains protective  antibodies  in  fairly  high  concentration  and  in  the 
case  of  females  the  immunity  is  transferred  to  the  offspring.  The 
quantity  of  antibodies  produced  by  this  method  of  immunization 
probably  is  in  proportion  to  the  severity  of  the  febrile  reaction 
which  follows  the  administration  of  the  immunizing  dose.  How- 
ever, strong  immunity  has  resulted  in  some  instances  in  which 
the  immunizing  injection  caused  only  a  barely  perceptible  febrile 
reaction. 

It  may  be  possible  to  use  the  immune  serum  from  the  horse, 
which  we  have  shown  to  be  susceptible  to  inoculation,  for  the 
prevention  of  the  disease  in  man.  For  this  purpose  the  serum 
should  be  injected  in  sufficient  quantity  within  two  or  three  days 
following  the  bite  of  the  tick.  Such  an  injection  should  not  be 
considered  protective  for  a  longer  period  than  three  weeks. 

The  method  of  sero-vaccination  is  not  yet  sufficiently  perfected 
to  warrant  its  application  to  man,  but  the  subject  is  being  studied 
further  in  order  to  determine  its  safety  and  efficiency. 

There  is  no  hope  of  obtaining  a  stronger  serum  for  curative 
purposes  than  that  yielded  by  an  animal  which  has  recently  recov- 
ered from  the  disease,  until  the  micro-organism  can  be  cultivated 
artificially,  thus  making  available  a  desirable  quantity  of  antigens 
for  immunization.  Even  with  this  condition  reaUzed  the  results 
of  further  immunization  cannot  be  anticipated  but  must  await 
experimental  determination. 


A    MICRO-ORGANISM    WHICH    APPARENTLY    HAS    A 

SPECIFIC  RELATIONSHIP  TO  ROCKY  MOUNTAIN 

SPOTTED  FEVER. 

A  PRELIMINARY  REPORT.' 

H.    T.    RiCKETTS. 
(From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

Since  the  spring  and  summer  of  1906,  bodies  which  I  have 
referred  to  in  my  notes  as  "  diplococcoid  bodies,"  and  sometimes 
short  bacillary  forms,  have  been  found  with  considerable  constancy 
in  the  blood  of  guinea-pigs  and  monkeys  which  were  infected  with 
Rocky  Mountain  spotted  fever.  They  have  also  been  seen  in  the 
blood  of  man  but  not  so  frequently.  Much  more  time  has  been 
spent  on  the  blood  of  the  experimental  animals  than  on  that  of 
man  in  view  of  the  fact  that  it  could  always  be  obtained  in  fresh  con- 
dition. 

The  form  most  commonly  found  is  that  of  two  somewhat 
lanceolate  chromatin-staining  bodies,  separated  by  a  slight  amount 
of  eosin-staining  substance.  The  preparation  of  Giemsa,  as  fur- 
nished by  Griibler,  has  been  used  almost  exclusively,  and  with 
variations  in  the  technic  the  intermediate  substance  may  stain 
faintly  blue. 

In  spite  of  the  constancy  with  which  these  bodies  were  found, 
it  did  not  seem  justifiable  to  claim  that  they  represent  the  micro- 
parasite  of  the  disease,  for  two  reasons:  (i)  the  very  complex 
morphology  of  the  blood,  especially  in  febrile  states,  when  various 
cells  and  probably  their  nuclei  are  subject  to  unusual  disintegra- 
tion; (2)  because  of  my  inability  to  cultivate  a  micro-organism 
of  this  character  from  infected  blood  by  the  use  of  ordinary  and 
some  unusual  culture  media,  under  various  conditions  of  cultivation, 
or  by  other  means  to  obtain  it  in  satisfactory  concentration.* 

'  From  Jour.  Am.  Med.  Assn.,  1909,  52,  p.  379.  See  also  " DemoQstration  of  a  Micro-organism  Which 
Apparently  Has  a  Specific  Relationship  to  Rocky  Mountain  Spotted  Fever,"  Trans.  Chic.  Path.  Soc, 
1907-9,  7,  p.  2S4- 

'  Mr.  P.  G.  Heinemann  assisted  in  an  extensive  series  of  culture  experiments  in  the  spring  of  1907. 

368 


A  Micro-organism  in  Spotted  Fever  369 

THE  BACILLUS   IN  THE   TICK. 

Although  infected  ticks  had  been  examined  previously  in  a  more 
or  less  cursory  manner,  their  systematic  study  was  not  undertaken 
until  recently.  In  the  pursuit  of  this  work,  advantage  was  taken 
of  the  fact  that  the  disease  is  transmitted  by  the  infected  female 
to  her  young  through  the  eggs,  as  described  in  a  previous  report. 
A  repetition  of  these  experiments  in  the  winter  of  1907-8,  with  the 
help  of  Dr.  Maria  B.  Maver,  resulted  in  such  transmission  in  50  per 
cent  of  the  ticks  used,  the  fact  being  determined  by  allowing  the 
larvae  to  feed  on  normal  guinea-pigs.  This  second  series  has  not 
been  published  heretofore. 

Female  tick  40,  a  dermacentor,  from  Montana,  had  produced 
fatal  infections  of  spotted  fever  in  guinea-pigs  1740  and  1764. 
A  number  of  eggs  from  the  first  day's  laying  were  crushed  in- 
dividually on  cover  glasses,  fixed  in  absolute  alcohol,  and  stained 
with  Giemsa's  stain.  Each  egg  was  found  to  be  laden  with  aston- 
ishing numbers  of  an  organism  which  appears  typically  as  a  bipolar 
staining  bacillus  of  minute  size,  approximating  that  of  the  influenza 
bacillus,  although  definite  measurements  have  not  yet  been  made. 
Various  forms  are  seen  depending  on  the  stage  of  development  and 
the  arrangement  in  which  two  or  more  may  be  found.  It  is  very 
common  to  find  two  organisms  end  to  end,  with  their  poles  stained 
deeply  and  the  intermediate  substance  a  faint  blue,  resembling  a 
chain  of  four  cocci.  When  the  chromatin  is  not  yet  sharply 
limited  to  the  poles,  the  somewhat  lanceolate  forms  so  often 
recognized  in  the  blood  are  seen.  Not  infrequently  delicate  bacilli 
with  a  uniform  distribution  of  the  chromatin  are  found.  These 
are  all  interpreted  as  stages  in  the  evolution  of  a  bipolar  organism. 
They  are  present  in  varying  numbers  in  different  eggs,  but  as  a 
rule  they  are  surprisingly  numerous,  and  in  some  instances  they 
would  certainly  count  into  the  thousands.  Many  faintly  staining, 
apparently  degenerate,  forms  are  encountered. 

Examination  of  the  eggs  of  three  dermacentors  from  Idaho 
(different  specifically  from  the  Montana  dermacentor),  which  were 
infected  from  the  guinea-pig,  showed  the  presence  of  the  same 
forms  (ticks  5,  7,  and  9). 


370  Contributions  to  Medical  Science 

Conference  with  zoologic  scientists  who  have  made  a  particular 
study  of  the  structure  of  eggs  brought  out  the  fact  that  such  bodies 
are  not  known  as  a  constituent  of  the  egg  of  any  species  of  animal. 

Although  it  has  not  yet  been  possible  to  examine  the  eggs  of 
ticks  which  are  known  to  be  free  from  spotted  fever,  the  equivalent 
control  has  been  made  through  a  comparison  of  the  visceral  organs 
of  infected  and  uninfected  ticks.  The  salivary  glands,  alimentary 
sac,  and  ovaries  of  infected  females  are  Hterally  swarming  with 
exactly  similar  micro-organisms.  On  the  contrary,  they  appear 
to  be  entirely  absent  from  the  viscera  of  the  uninfected  tick,  both 
male  and  female. 

agglutination  reactions. 

The  most  striking  evidence  of  the  probable  etiologic  relationship 
of  this  organism  to  spotted  fever  is  found  in  the  positive  outcome 
of  agglutination  tests.  Fortunately  the  organism  is  so  numerous 
in  the  eggs  that  a  bacterial  emulsion  of  reasonable  concentration 
for  agglutination  tests  can  be  made  by  crushing  forty  to  fifty  eggs 
in  about  0.05  c.c.  of  salt  solution.  The  material  is  so  scant  that 
only  the  microscopic  method  could  be  used.  The  preparations 
were  made  as  hanging  drops,  incubated  for  two  hours,  dried,  fixed 
with  absolute  alcohol,  and  stained. 

The  serum  of  the  normal  guinea-pig  either  causes  no  agglutina- 
tion at  all,  or  at  the  most  produces  only  sHght  agglutination  in 
proportions  of  i  to  i  and  i  to  20.  Dilutions  higher  than  this 
cause  no  agglutination.  In  testing  the  agglutinating  powers  of 
immune  serums,  three  animals  which  had  been  infected  from 
different  sources  and  had  recovered  were  used.  One  (1751)  had 
been  infected  with  a  dermacentor  from  Idaho;  another  (1692), 
with  a  strain  handed  down  direct  from  guinea-pig  to  guinea-pig  for 
nearly  three  years  without  the  intervention  of  ticks,  the  original 
infection  having  been  obtained  from  the  blood  of  man;  the  third 
(1757),  with  a  strain  kept  in  the  same  way  since  last  spring.  Graded 
dilutions,  beginning  with  i  to  i  and  going  as  high  as  i  to  400,  were 
used  in  the  different  series,  with  the  striking  result  that  a  complete 
agglutinating  power  was  present  in  the  three  immune  serums  in 
dilutions  up  to  i  to  320.     It  was  somewhat  less  in  a  dilution  of  i 


A  Micro-organism  in  Spotted  Fever  371 

to  400.  The  highest  dilution  which  will  cause  clumping  has  not 
been  ascertained. 

No  fresh  immune  serum  from  man  is  at  hand,  but  tests  were 
made  with  three  specimens  which  are  about  five,  seven,  and  nine 
months  old,  respectively.  They  have  been  preserved  in  the  ice- 
chest  with  the  addition  of  0.3  per  cent  of  chloroform.  The  pecul- 
iar phenomenon  of  failure  to  agglutinate  in  concentrated  solution 
was  noted  with  all  three.  With  the  oldest  serum  no  agglutination 
occurred  until  the  dilutions  of  i  to  320  and  i  to  400  were  reached, 
when  incomplete  clumping  was  produced.  With  the  second  there 
was  no  agglutination  in  the  dilution  of  i  to  i,  distinct  clumping 
in  I  to  20, 1  to  40,  and  i  to  80,  with  httle  or  none  in  higher  dilutions. 
In  the  serum  of  five  months  standing  the  reaction  was  absent 
in  the  dilutions  of  i  to  i,  i  to  20,  and  i  to  40,  positive  but  not  com- 
plete in  I  to  80,  I  to  160,  and  i  to  240,  with  very  little  clumping 
in  I  to  320  and  i  to  400.  Normal  human  serum  caused  clumping 
in  a  dilution  of  i  to  i,  a  very  slight  amount  in  i  to  20,  and  none  at 
all  in  the  higher  dilutions.^ 

The  failure  of  the  immune  serums  from  man  to  agglutinate  in 
concentrated  solutions,  whereas  they  did  so  in  higher  dilutions,  is 
taken  as  an  example  of  the  action  of  the  so-called  proagglutinoids. 
As  explained  by  Ehrlich's  theory,  this  consists  in  the  occupation 
of  the  bacterial  receptors  by  inactive  agglutinin  which  exceeds  the 
active  agglutinin  in  its  affinity  for  the  bacteria.  With  higher 
dilutions  of  the  serum  the  agglutinoids  are  so  diluted  that  they  do 
not  completely  occupy  the  bacterial  receptors,  thus  affording  a 
point  of  attack  for  the  active  agglutinin,  unless  the  latter  has  been 
eliminated  by  extreme  dilution.  This  is  well  known  as  a  property 
of  old  agglutinating  serums. 

bacilli  in  infected  serum. 

As  a  means  of  concentrating  the  organisms  in  the  serum  of  the 
infected  guinea-pig  the  following  experiment  was  performed: 
Three  cubic  centimeters  of  fresh  infected  serum  were  diluted  with 
an  equal  amount  of  salt  solution,  and  to  this  was  added  0.3  c.c. 
of  an  agglutinating  serum  from  the  guinea-pig.     The  mixture  was 

'  Some  of  the  dilutions  were  wrongly  stated  in  The  Journal.    They  have  been  corrected  in  this  reprint. 


372  Contributions  to  Medical  Science 

placed  in  the  incubator  for  two  hours  and  then  centrifugated  for 
about  ten  hours  at  a  speed  of  i,8oo  revolutions.  All  but  the  merest 
drop  was  then  pipetted  off  and  stained  preparations  were  made 
of  the  sediment.  Examination  showed  the  presence  of  a  moderate 
number  of  forms  which  are  identical  in  appearance  and  size  with 
those  often  recognized  in  ordinary  smears  of  infected  blood,  and 
also  with  the  "  diplococcoid "  forms  seen  in  the  egg  of  the  infected 
tick.     No  such  bodies  were  found  in  a  control  tube  of  normal  serum. 

The  evidence  pointing  to  this  organism  as  the  causative  agent 
in  spotted  fever,  though  not  complete,  is  of  a  striking  character. 
In  so  far  as  I  know  it  would  be  an  unheard-of  circumstance  to  obtain 
such  strong  agglutination  with  an  immune  serum,  in  the  presence 
of  negative  controls,  unless  there  were  a  specific  relationship 
between  the  organism  and  the  disease.  In  favor  of  the  specific 
relationship  in  this  case  are  also  the  presence  of  the  organism  in 
large  numbers  in  infected  ticks  and  in  their  eggs,  its  absence  from 
uninfected  ticks,  and  the  presence  of  similar  forms  in  the  blood  and 
serum  of  the  infected  guinea-pig. 

Morphologically  the  organism  is  a  bacillus  and  somewhat 
pleomorphic  as  described.  Its  resemblance  to  the  bacilli  of  the 
hemorrhagic  septicemias  is  striking,  and  in  this  connection  it  is 
important  to  note  that  spotted  fever  is  a  hemorrhagic  septicemia. 
It  has  not  been  cultivated,  although  work  with  this  end  in  view 
is  in  progress. 

I  have  devised  no  formal  name  for  the  organism  discussed,  but 
it  may  be  referred  to  tentatively  as  the  bacillus  of  Rocky  Mountain 
spotted  fever.  A  further  study  of  its  characteristics  may  suggest  a 
suitable  name. 

That  a  bacillus  may  be  the  causative  agent  of  a  disease  in  which 
an  insect  carrier  plays  an  obhgate  role  under  natural  conditions 
may  be  looked  at  with  suspicion  in  some  quarters.  Yet,  even  with- 
out the  evidence  in  this  case,  it  would  seem  unscientific  to  be 
tied  to  the  more  or  less  prevailing  belief  that  all  such  diseases  must, 
on  the  basis  of  several  analogies,  be  caused  by  parasites  which  are 
protozoan  in  character. 

Further  study  of  the  relationship  of  the  bacillus  to  the  disease 
is  being  carried  on  and  will  be  reported  at  a  future  date,  together 
with  illustrations  and  a  more  detailed  account  of  its  characteristics. 


SOME     ASPECTS     OF     ROCKY     MOUNTAIN     SPOTTED 
FEVER  AS  SHOWN  BY  RECENT  INVESTIGATIONS. 

THE    WESLEY    M.     CARPENTER     LECTURE    OF    THE    NEW    YORK 
ACADEMY  OF  MEDICINE,   1909.* 

H.     T.     RiCKETTS. 

As  your  lecturer  on  this  occasion  it  will  be  my  privilege,  a  most 
honorable  one,  to  present  a  more  or  less  succinct  account  of  an 
investigation  of  Rocky  Mountain  spotted  (or  tick)  fever,  which  has 
been  under  way  for  more  than  three  years,  and  which,  it  is  believed, 
has  contributed  results  of  value  for  a  better  understanding  of  the 
disease  in  certain  important  aspects.  Many  of  the  essentials  have 
been  described  in  scattered  articles  in  two  different  journals,  and 
in  two  reports  which  have  been  rendered  to  the  State  Board  of 
Health  of  Montana.  However,  such  a  presentation  as  I  wish  to 
give,  involving  some  new  data,  a  consideration  of  the  natural  his- 
tory of  the  disease,  and  certain  observations  regarding  its  microbic 
etiology,  may  perhaps  free  me  from  a  possible  charge  of  too  much 
repetition. 

History  and  occurrence. — It  would  be  interesting  if  one  were  able 
to  give  a  satisfactory  history  of  the  disease,  bearing  on  its  first 
appearance  among  the  native  Indians  and  among  the  white  men 
of  the  different  "infected  districts,"  its  early  geographic  distri- 
bution, and  the  possibility  of  its  having  extended  into  new  terri- 
tories. Knowledge  on  these  points  is  very  incomplete,  however, 
and  regarding  its  antiquity  it  can  only  be  stated  that  it  was  a  well- 
known  disease  when  certain  of  the  older  physicians  and  residents 
first  came  into  the  valley  of  the  Snake  River,  in  southern  Idaho, 
thirty  or  forty  years  ago.  In  the  Bitter  Root  Valley  in  western 
Montana  it  is  difficult  or  impossible  to  learn  definitely  of  cases  which 
appeared  prior  to  the  early  '8o's.  Hence  it  is  possible,  though  by  no 
means  to  be  taken  for  granted,  that  spotted  fever  appeared  more 
recently  in  western  Montana  than  in  Idaho.  The  Bitter  Root  Valley 
is  separated  from  Idaho  only  by  the  Bitter  Root  range  of  mountains, 

*  From  Med.  Record,  1909,  76,  p.  842. 

373 


374  Contributions  to  Medical  Science 

and  since  spotted  fever  occurs  only  on  the  west  slope  of  the  Bitter 
Root  Valley,  it  is  conceivable  that  the  disease  extended  into  the 
latter  region  from  Idaho  through  the  various  mountain  passes 
and  that  it  has  as  yet  failed  to  reach  the  eastern  slope  of  the  valley. 
It  is  known  as  particularly  hazardous  to  visit  certain  canyons  of  the 
Bitter  Root  Mountains  during  the  months  of  spring. 

Its  distribution  in  any  one  state  appears  to  be  very  irregular, 
although  it  is  found  here  and  there  over  a  wide  range  of  territory; 
in  southern  Idaho  along  the  course  of  the  Snake  River,  and  in  west- 
ern Montana  as  stated,  at  another  point  in  eastern  Montana,  near 
Bridger,  in  the  northern  and  northwestern  parts  of  Wyoming 
south  of  the  Bridger  district,  in  northern  Utah,  eastern  Oregon, 
and  in  ill-defined  districts  of  Colorado  and  Nevada.  In  none  of 
these  states,  however,  except  Idaho  and  Montana,  has  any  system- 
atic attempt  been  made  to  learn  the  exact  occurrence  of  the 
disease. 

The  irregular  geographic  distribution  is  greatly  accentuated 
in  certain  instances.  Thus,  in  Idaho,  for  many  years  the  disease 
was  known  to  occur  only  on  the  north  slope  of  the  Snake  River 
Valley,  and  if  my  informants  are  correct,  it  has  existed  on  the 
south  side  of  the  valley,  in  the  eastern  part  of  the  state,  only  during 
the  past  twelve  or  fifteen  years.  Its  limitation  to  the  west  slope 
of  the  Bitter  Root  Valley  was  mentioned.  The  various  possibihties 
which  may  be  adduced  in  the  explanation  of  this  peculiarity  and 
the  factors  which  operate  for  extension,  on  the  one  hand,  and  for 
its  limitation  on  the  other,  cannot  well  be  discussed  within  the 
limits  of  this  lecture. 

No  satisfactory  estimate  can  be  given  of  the  number  of  cases 
which  appear  annually  over  this  whole  territory.  In  the  two 
infected  districts  of  Montana  the  number  probably  varies  from 
fifteen  to  thirty.  Twelve  occurred  in  the  Bitter  Root  Valley  in 
1908  and  twenty-six  or  twenty-eight  in  1909.  The  increased  num- 
ber during  the  past  spring  was  due  to  railroad  construction,  which 
resulted  in  the  exposure  of  an  unusual  number  of  persons.  In 
1908,  Dr.  Edward  E.  Maxey,  of  Boise,  Idaho,  did  an  admirable 
piece  of  work,  in  which  he  obtained  all  possible  data  regarding 
spotted  fever  from  the  physicians  of  the  state,  locating  the  disease 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    375 

exclusively  in  the  basin  of  the  Snake  River,  chiefly  on  the  north 
slope  of  the  valley;  380  cases  occurred  in  Idaho  during  that  year.' 
The  work  of  Dr.  Maxey  urgently  demands  repetition  in  other  states. 

There  is  no  more  striking  feature  of  the  disease  than  its  sharp 
limitation  to  the  months  of  the  spring.  In  Montana  it  makes 
its  first  appearance  in  the  latter  part  of  March  or  April,  and  the 
last  cases  are  seen  about  the  middle  of  June.  The  spring  is  earlier 
in  southern  Idaho  and,  corresponding  with  this,  the  "spotted  fever 
season"  begins  and  closes  from  two  to  four  weeks  earlier  than 
in  Montana.  The  relation  of  the  tick  to  this  peculiar  seasonal 
occurrence  will  be  referred  to  again. 

General  features. — Spotted  fever  has  a  history  in  literature  since 
1899,  when  Maxey  defined  it  as  follows:  "  an  acute  endemic, 
non-contagious,  but  probably  infectious,  febrile  disease,  character- 
ized clinically  by  a  continuous  moderately  high  fever,  severe  ar- 
thritic and  muscular  pains,  and  a  profuse  petechial  or  purpural 
eruption  in  the  skin,  appearing  first  on  the  ankles,  wrists,  and  fore- 
head, but  rapidly  spreading  to  all  parts  of  the  body."^  I  shall 
not  at  this  time  discuss  in  detail  the  various  clinical  aspects  of  the 
disease,  but  rather  refer  you  to  the  reports  of  Maxey ,^  Wilson 
and  Chowning,-5  Anderson,-*  and  Stiles^  for  information  in  this 
regard.  I  may  only  state  that,  in  my  experience,  variations  from 
the  typical  type  of  eruption  are  encountered  rather  frequently.^ 

The  anatomical  changes  are  not  highly  characteristic  with  the 
exception  of  the  cutaneous  phenomena  which  have  been  mentioned. 
Gangrene  of  the  scrotum,  prepuce,  faucial  pillars,  and  soft  palate 
sometimes  occur.  Strangely  this  is  met  with  more  frequently  in 
Idaho  than  in  Montana,  and  the  explanation  may  lie  in  the  differ- 
ence in  the  duration  of  the  infection  in  the  two  localities.  Cus- 
tomarily patients  in  Montana  die  before  the  period  when  gangrene 
is  likely  to  occur,  whereas  in  Idaho  the  large  percentage  of  recov- 
eries gives  sufficient  opportunity  for  the  development  of  this 
phenomenon.  Gangrene  in  both  man  and  animals  is  a  compara- 
tively late  symptom.  The  most  striking  and  constant  visceral 
change  is  found  in  the  spleen,  which  is  always  enlarged  and  may. 
weigh  two  or  three  times  as  much  as  the  normal  organ.  Its  con- 
sistence is  rather  firm  and  gives  the  appearance  of  a  very  cellular 


376  Contributions  to  Medical  Science 

organ,  such  as  might  be  caused  by  a  massive  proliferation  of  pulp 
and  endothelial  cells.  It  has  none  of  the  soft  semi-gelatinous 
appearance  of  the  typhoid  spleen.  The  lymphatic  glands  are 
uniformly  enlarged  and  moderately  congested,  but  in  man  I  have 
not  found  them  hemorrhagic.  The  kidneys  are  swollen  and 
degenerated  and  the  liver  is  moderately  enlarged,  congested,  and 
may  be  fatty.  No  striking  or  constant  changes  are  found  in  the 
suprarenals,  heart,  and  lungs.  The  central  nervous  system  shows 
no  alterations  except  slight  meningeal  congestion  and  edema,  but 
these  are  of  a  very  minor  order.  In  one  case  I  found  a  lobular 
pneumonia  and  the  appearance  of  multiple  infarctions  of  the  spleen. 
The  conditions  were  such  that  the  bacteriology  of  the  case  could 
not  be  worked  out. 

The  microscopic  anatomy  is  in  the  hands  of  Professor  E.  R. 
Le  Coimt. 

Severe  and  mild  types. — ^As  regards  severity,  two  different  types 
are  to  be  recognized,  the  mild  and  the  severe,  and  in  this  point 
lies  another  of  the  pecuHarities  of  the  disease.  In  western  Mon- 
tana it  is  almost  uniformly  of  a  very  severe  character,  the  mor- 
tality ranging  from  about  65  per  cent  to  about  90  per  cent. 
Children  recover  with  some  frequency,  adults  very  rarely.  In  Idaho, 
on  the  other  hand,  the  death  rate  rarely  rises  above  5  per  cent, 
and  in  the  380  cases  which  Maxey  collected  in  1908  it  was  4.86 
per  cent  and  3 . 8  per  cent  for  the  preceding  year.  This  difference 
would  not  be  so  remarkable  were  it  not  constant;  the  spotted 
fever  of  Montana  has  always  been  severe,  while  that  of  Idaho  has 
always  been  mild.  Occasional  cases  in  the  latter  state  go  through 
the  entire  course  of  fever  without  being  confined  steadily  in  bed. 
This  feature  suggests  that  the  disease  in  the  two  localities  may  not 
be  absolutely  identical,  but  may  rather  represent  varieties,  just  as 
we  accept  paratyphoid  fever  as  a  variety  of  typhoid. 

The  difference  in  virulence  is  also  borne  out  by  animal  inocula- 
tions. Guinea-pigs  may  be  infected  with  the  Idaho  disease  by  the 
injection  of  diseased  human  blood,  just  as  in  the  case  of  the  Montana 
disease,  and  the  cHnical  and  anatomical  phenomena  are  the  same 
in  both  cases.  The  Montana  disease  may  be  kept  going  indefi- 
nitely in  the  guinea-pig  by  consecutive  passage  with  a  high  rate 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    377 

of  mortality  (about  90  per  cent).  On  three  occasions,  however, 
and  with  material  taken  from  different  patients,  I  have  been  unable 
to  maintain  the  Idaho  disease  in  the  same  way.  In  one  instance 
it  was  carried  into  the  third  passage ;  in  another,  into  the  fourth ; 
and  in  the  third,  into  the  tenth. 

In  spite  of  this  constant  difference,  their  practical  identity  is 
shown  not  only  by  the  identity  of  the  clinical  manifestations,  but 
also  by  specific  immunity  tests.  Guinea-pigs  which  have  recovered 
from  the  Idaho  disease  are  immune  to  that  of  Montana,  and  the 
converse  is  also  true.  It  may  be  that  quantitative  determinations 
of  the  protective  and  agglutinating  power  of  the  two  immune 
serums  will  show  such  differences  that  the  two  diseases  may  be 
considered  to  stand  in  a  sort  of  "group  relationship"  to  each  other. 

Although  a  satisfactory  explanation  of  this  difference  may  not 
be  possible,  it  is  of  interest  to  note  that  the  spotted  fever  tick 
of  Idaho  differs  specifically  from  that  of  Montana.  Banks  has 
identified  the  latter  as  Dermacentor  venustus.  The  Idaho  tick 
is  also  a  dermacentor,  but  its  markings  are  much  less  distinct  than 
those  of  D.  venustus,  and  it  is  constantly  smaller  in  size  in  all  its 
stages,  as  shown  by  numerous  comparative  measurements  of  the 
eggs,  larvae,  nymphs,  and  adults.  From  specimens  sent  to  Banks, 
he  concluded  that  the  species  had  not  been  described  previously, 
and  he  gave  it  the  name  of  Dermacentor  modestus.  Our  experi- 
ments have  shown  that  both  venustus  and  modestus  will  transmit 
the  Montana  disease  to  the  guinea-pig,  and  venustus  will  also 
transmit  the  Idaho  disease.  So  far,  we  have  been  unable  to  obtain 
transmission  of  the  Idaho  disease  to  the  guinea-pig  by  the  use  of  the 
Idaho  tick,  i.e.,  D.  modestus,  except  in  one  instance  in  which  a 
large  number  of  ticks  was  involved.  It  will  be  for  future  experi- 
ments to  determine  whether  this  is  due  to  the  comparatively  low 
virulence  of  the  Idaho  virus  for  the  guinea-pig,  as  it  exists  in  D. 
modestus;  in  other  words,  whether  a  larger  amount  of  this  strain 
is  required  for  the  infection  of  the  guinea-pig  than  a  single  tick  of 
this  species  is  able  to  inject  during  its  feeding. 

It  is,  of  course,  possible  that  the  tick  plays  only  a  partial  role 
in  the  maintenance  of  virulence.  As  indicated  later,  the  disease 
appears  to  be  maintained  in  nature  by  alternating  between  the 


378  Contributions  to  Medical  Science 

tick  and  some  one  or  more  of  the  native  small  animals.  It  is 
possible  that  the  animals  which  play  this  part  in  Idaho  are  not 
identical  with  those  enacting  a  similar  role  in  Montana,  and  if 
this  proves  to  be  the  case,  the  difference  in  virulence  may  depend 
in  whole  or  in  part  on  the  difference  in  the  animals  which  serve 
as  hosts  in  the  two  localities.  This  may  or  may  not  be  open  to 
experimental  determination. 

Theories  regarding  the  source  of  injection. — In  the  past  various 
local  theories  have  prevailed,  and  do  still  prevail  to  a  certain 
extent,  regarding  the  source  of  infection  for  man.  The  sharp 
limitation  of  spotted  fever  to  the  months  of  spring,  suggested  to 
the  residents  and  to  many  of  the  physicians  that  the  virus  may  be 
obtained  from  the  water  which  flows  down  from  the  mountains  dur- 
ing the  melting  of  the  snow.  It  was  assumed  that  this  resulted  in 
the  flushing  out  of  certain  low  places  which  contained  decaying 
vegetation,  in  which  the  virus  or  poison  might  breed  or  have  its 
source.  In  the  Bitter  Root  Valley  in  Montana  so  many  of  the 
patients  were  employees  of  sawmills,  or  lived  in  the  vicinity  of  such 
mills,  that  the  disease  also  became  associated  with  this  industry. 
Old  and  decaying  accumulations  of  sawdust  were  supposed  to  con- 
taminate the  water,  and  many  patients  give  a  history  of  having 
drunk  water  from  a  stream  on  which  a  sawmill  previously  had  its 
site.  It  is  pointed  out  that  there  was  virtually  no  spotted  fever 
until  the  lumbering  industry  entered  the  valley. 

In  spite  of  a  fair  degree  of  plausibility  which  attached  to  the 
water  theory,  careful  examination  of  the  distribution  of  cases 
during  any  one  season  shows  that  this  does  not  correspond  to  any 
type  of  water  supply.  Indeed,  one  of  the  characteristic  features 
of  the  disease,  though  it  is  an  accidental  one,  is  the  rare  occurrence 
of  more  than  one  case  in  a  given  family  during  a  season,  and  the 
number  of  cases  which  occur  on  any  one  stream  during  a  season 
is  too  small  to  admit  of  the  water  theory.  Also  the  rough  coin- 
cidence of  the  spotted  fever  season  with  that  of  the  melting  snow 
and  high  water  is  a  misleading  one.  A  few  consecutive  warm  days 
are  commonly  followed  by  a  crop  of  cases  of  spotted  fever  in  the 
spring.  This,  of  course,  does  melt  more  or  less  snow,  and  does 
cause  a  rise  in  the  streams,  but  it  is  equally  efficient  in  rousing 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    379 

from  lethargy  millions  of  ticks  which  in  cooler  weather  are  too 
somnolent  to  respond  to  the  presence  of  a  host. 

The  lumbering  industry  has  indeed  been  a  factor,  but  only  in 
this,  that  it  took  its  employees  into  the  timbered  country  which 
harbors  infected  ticks,  and  caused  a  general  increase  in  the  popu- 
lation of  the  valley,  with  the  consequent  exposure  of  a  larger  num- 
ber of  persons.  The  industry  of  railroad  construction  did  the  same 
thing  in  1909.  Furthermore,  there  is  no  lumbering  in  southern 
Idaho,  where  spotted  fever  is  much  more  common  than  in  Montana. 

The  theory  that  the  tick  may  have  a  relationship  to  spotted 
fever  had  a  fluctuating  existence  in  both  Montana  and  Idaho  for 
some  time,  but  it  was  not  generally  credited,  and  really  had  its 
birth  with  the  publication  of  Wilson  and  Chowning  in  1902. 

Previous  investigations. — A  laboratory  study  of  the  disease  was 
first  undertaken  in  1902-3  by  Dr.  Louis  B.  Wilson  and  Dr.  William 
M.  Chowning,  then  of  the  University  of  Minnesota.''  In  addition 
to  giving  an  account  of  the  clinical  and  anatomical  phases  of  the 
malady,  they  advanced  the  hypothesis  that  the  tick  is  the  means 
of  infecting  man;  described  as  the  cause  a  protozoan  parasite,  to 
which  they  gave  the  name  of  Piroplasma  hominis,  and  which  they 
found  to  have  a  specific  location  within  the  erythrocytes;  and 
suggested  that  the  ground  squirrel  may  be  the  source  of  infection 
for  the  ticks,  basing  this  hypothesis  on  the  supposed  presence  of 
the  same  parasite  in  the  erythrocytes. 

Their  work  was  followed  by  that  of  several  members  of  the 
Public  Health  and  Marine  Hospital  Service  during  succeeding 
years:  Anderson,  Stiles,  and  King.  Anderson  agreed  with  the 
essential  claim  of  Wilson  and  Chowning,  namely,  the  existence 
of  a  piroplasma  in  the  erythrocytes  of  the  patients,  and  also  took  a 
stand  favoring  the  hypothesis  of  transmission  by  the  tick.  Stiles, 
on  the  other  hand,  was  unable  to  find  the  piroplasma,  and  expressed 
his  disbelief  in  the  tick  theory.  Wilson  and  Chowning  had  no 
experimental  proof  of  the  correctness  of  this  theory,  just  as  Stiles 
had  none  to  show  its  incorrectness. 

Personal  studies. — The  investigations  of  which  it  is  the  purpose 
to  speak  were  inaugurated  in  the  spring  of  1906  with  the  discovery 
that  the  guinea-pig  and  monkey  may  be  infected  with  spotted  fever 


380  Contributions  to  Medical  Science 

through  the  subcutaneous  or  intraperitoneal  injection  of  the  blood 
of  human  patients.*  In  view  of  the  fact  that  the  disease  has  been 
without  a  known  bacteriology  during  the  course  of  the  work,  it 
has  been  necessary  to  rely  on  the  cHnical  course  and  anatomical 
changes  in  the  guinea-pig,  together  with  negative  bacteriological 
findings,  as  criteria  for  the  recognition  of  the  disease  in  this  animal. 
Since  the  reliability  of  the  different  phases  of  the  work  really 
depends  on  this  point,  it  is  desirable  to  emphasize  that  spotted 
fever  in  the  guinea-pig  is  a  characteristic  condition,  and  that  con- 
fusion can  occur  but  rarely. 

Spotted  fever  in  the  guinea-pig. — When  the  inoculation  is  accom- 
plished by  the  injection  of  virus  (diseased  blood,  and  the  organs  or 
eggs  of  infected  ticks),  an  incubation  period  of  from  two  to  five 
days  is  followed  by  a  sudden  rise  in  temperature,  which  may  reach 
105°  or  106°  F.  (in  rare  instances  107°)  within  two  or  three  days, 
and  which  persists  without  remission  for  a  period  of  from  six  to 
twelve  days,  or  until  the  death  of  the  animal,  four  to  ten  or  twelve 
days  after  onset.  A  swelling  of  the  scrotum,  which  begins  in  the 
males  on  the  third  or  fourth  day  of  fever,  progresses  rapidly,  and 
the  overlying  skin  is  soon  infiltrated  with  blood.  In  white-skinned 
animals,  which  are  shaved  at  this  time  or  later,  a  generalized  roseo- 
lar  eruption,  most  marked  on  the  back,  extremities,  and  face,  can 
be  made  out  readily.  In  females  the  vulva  becomes  swollen,  but 
exhibits  hemorrhages  only  in  occasional  cases.  Swelling,  conges- 
tion, and  hemorrhage  of  the  lymphatic  glands,  a  more  or  less  massive 
enlargement  of  the  spleen,  which  is  cyanotic  in  color,  and,  in  males, 
marked  congestion  of  the  tunica  vaginalis  and  often  of  the  testicles 
are  the  essential  anatomical  changes,  which  are  encountered  with 
little  variation.  Commonly  the  kidneys  are  swollen  and  degen- 
erated, the  Hver  swollen,  and  the  suprarenals  may  show  cortical 
hemorrhages.  The  serous  surfaces,  lungs,  heart,  and  central 
nervous  system  are  never  involved,  in  the  absence  of  mixed  infec- 
tions. As  indicated  by  ordinary  culture  media,  the  blood  and  vis- 
cera are  sterile. 

*  The  monkey  (Macacus  rhesus),  rabbit,  horse,  and  at  least  five  species  of  small  wild  animals  have  a 
greater  or  less  degree  of  susceptibility.  In  aU  of  these  except  the  monkey  confirmatory  transfers  must  be 
made  into  the  guinea-pig  in  order  to  prove  the  character  of  the  infection;  hence,  for  this  reason  among 
others,  they  are  of  no  service  for  routine  experimentation. 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    381 

The  "immunity  test"  is  an  important  and  sometimes  an  essen- 
tial aid  to  diagnosis  in  animals  which  recover.  In  the  event  of 
mild  infection,  which  has  occurred  accidentally,  or  which  has  been 
produced  deliberately,  by  modified  virus,  the  fever  may  be  short 
in  duration  and  moderate  in  height,  and  the  external  hemorrhages 
may  not  appear.*  In  such  cases  the  immunity  test,  which  consists 
of  the  injection  of  from  500  to  1,000  pathogenic  doses  of  infected 
blood,  determines  whether  or  not  spotted  fever  was  present  in  the 
first  instance.  This  active  immunity  is  strong  and  of  long  dura- 
tion, and  the  offspring  of  immune  females  possess  a  passive  im- 
munity which  persists  for  a  period  of  from  three  to  four  months. 

These  data  have  proved  sufficient  for  the  positive  recognition 
of  spotted  fever  in  the  guinea-pig,  and  on  three  occasions  they  have 
enabled  us  to  recognize  mixed  infections  which  crept  into  experi- 
ment animals  in  some  unknown  way. 

The  transfer  of  spotted  fever  to  the  guinea-pig  from  human 
patients  in  Montana  and  Idaho  has  now  been  repeated  not  less 
than  fifteen  times  from  as  many  different  patients,  and  with 
practically  unvarying  results.  The  oldest  strain  now  on  hand, 
representing  the  Montana  disease,  has  been  kept  alive  by  passage 
in  the  guinea-pig  for  three  and  one-half  years,  with  symptoms  and 
anatomical  changes  which  do  not  vary  from  those  produced  in  the 
first  animals  injected,  and  with  no  decrease  in  the  virulence  of  the 
diseased  blood.  Two  other  "  human  "  strains  have  been  maintained 
in  the  same  way,  each  for  approximately  a  year,  when  they  were 
dropped.  Also  a  strain  from  naturally  infected  ticks  (D.  venustus) 
has  now  been  maintained  for  six  months.  Inasmuch  as  the  work 
has  been  done  with  these  various  strains  more  or  less  indiscrimi- 
nately, it  is  important  to  be  certain  of  their  unity,  and  this  is  shown 
not  only  by  the  identity  of  the  cHnical  and  anatomical  phenomena, 
but  also  by  the  fact  that  each  establishes  immunity  against  the 
others.  The  integrity  of  the  infection  in  the  guinea-pig  was  also 
confirmed  by  attempts  at  reinoculation  with  fresh  human  virus; 
animals  which  had  recovered  from  inoculations  with  a  strain  grown 
in  the  guinea-pig  for  a  year  proved  immune  to  such  inoculations, 

•  In  some  successful  vaccination  experiments  a  moderate  rise  in  temperature  for  only  one  day  was  all 
that  marked  the  course  of  infection.    Such  animals  were  thereafter  immune. 


382  Contributions  to  Medical  Science 

and  from  the  further  correspondence  of  the  clinical  and  anatomical 
manifestations  in  man  and  the  guinea-pig,  we  may  be  certain  that 
the  infection  which  has  been  used  in  the  experimentation  is  identi- 
cal with  human  spotted  fever.  That  the  disease  is  an  infection 
rather  than  an  intoxication  by  non-living  material  is  abundantly 
demonstrated  by  the  statements  just  made;  the  causative  agent 
must  be  one  which  is  capable  of  proliferation,  in  order  to  survive 
two  hundred  consecutive  passages  in  the  guinea-pig  with  undi- 
minished virulence. 

Experiments  with  the  tick. — With  the  demonstration  of  the 
exquisite  susceptibility  of  the  guinea-pig  it  was  manifestly  possible 
to  proceed  to  an  investigation  of  the  hypothesis  of  transmission 
by  the  tick.* 

The  technic  of  this  work  was  very  crude  in  the  beginning,  but 
satisfactory  methods  were  soon  evolved,  as  described  in  a  prior 
communication.^  The  first  problem  was  one  of  simple  transmis- 
sion, namely,  is  the  tick  (D.  andersoni  of  Stiles,  D.  venustus  of 
Banks)  able  to  transmit  spotted  fever  from  the  sick  to  the  healthy 
animals  by  means  of  its  bite  ?  The  initial  successful  results, 
obtained  with  a  single  female  and  later  with  a  single  male  (1906), 
have  been  repeated,  I  should  estimate,  not  less  than  two  hundred 
times  in  the  course  of  various  experiments  conducted  in  the  field 
and  home  laboratories.  There  is,  indeed,  no  difficulty  in  obtain- 
ing it.  It  is  only  necessary  to  permit  a  tick  to  feed  on  a  diseased 
guinea-pig  for  several  hours  to  a  day,  and  then  to  transfer  the 
arachnid  to  a  healthy  animal  as  soon  as  it  can  again  be  induced  to 
bite. 

The  exact  duration  of  feeding  required  for  infection  of  the  tick 
has  not  been  determined.     This  must  vary  a  good  deal.     The  tick 

*  The  infective  dose  of  diseased  blood  drawn  on  the  third  day  of  fever  in  the  guinea-pig  is  frequently 
as  small  as  o.ooi  c.c,  when  injected  intraperitoneally  into  a  fresh  animal. 

It  is  interesting  to  note  that  the  first  experiments  which  were  performed  with  the  intention  of  deter- 
mining a  possible  relationship  of  the  tick  to  spotted  fever  were  done  on  the  human  subject,  and  have  not 
been  formally  reported  by  the  authors.  I  have  referred  to  this  in  a  former  article,  and  here  may  only  repeat 
that  Drs.  McCalla  and  Brereton,  of  Boise,  Idaho,  infected  two  individuals  successively  by  the  bite  of  a  tick 
which  they  had  removed  from  one  of  their  patients.  Naturally,  the  consent  of  the  subjects  was  obtained, 
which  in  this  case  was  not  difficult  on  account  of  the  mild  nature  of  the  disease  in  Idaho.  The  exf)eriments 
of  McCalla  and  Brereton  followed  the  publication  of  the  tick  hypothesis  by  Wilson  and  Chowning,  and 
they  were  unknown  to  me  until  after  the  larger  part  of  my  work  with  the  tick  had  been  done. 

The  first  transmissions  of  spotted  fever  in  the  guinea-pig  by  means  of  the  tick  were  undertaken  and 
reported  almost  simultaneously  by  Dr.  W.  W.  King,  of  the  Public  Health  and  Marine  Hospital  Service, 
and  by  the  writer. 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    ^8 


O'-'O 


is  a  slow  feeder.  From  fifteen  to  twenty  minutes  are  required  for 
the  hungriest  and  most  vigorous  to  attach  itself  firmly,  and  without 
firm  attachment  by  means  of  the  palpi,  feeding  is  impossible.  In 
other  instances  the  process  is  much  slower,  and  after  two  or  three 
days  of  attachment  there  is  little  evidence  of  the  ingestion  of  blood. 

In  so  far  as  we  have  determined,  the  incubation  period  in  the 
tick  is  brief,  and,  perhaps,  is  represented  only  by  the  time  required 
for  the  distribution  of  the  virulent  organisms  throughout  its  body 
and  eventually  into  its  salivary  glands.  In  a  number  of  instances 
the  tick  proved  virulent  immediately  following  its  removal  from  the 
infected  animal.  In  one  exceptional  experiment,  transmission 
could  not  be  obtained  until  nearly  two  weeks  following  the  infected 
feed. 

Likewise  the  minimum  duration  of  the  bite  required  for  inocu- 
lation probably  varies  a  good  deal,  depending  on  the  vigor  of  feed- 
ing. Dealing  with  ticks  whose  infection  had  already  been  proved 
by  longer  feedings,  transmission  resulted  several  times  from  ten- 
hour  exposures,  and  recently  from  a  feed  of  less  than  two  hours.* 

The  mechanism  of  transfer  would  seem  to  consist  of  the  injec- 
tion of  the  saHvary  secretion,  laden  with  virus,  into  the  cutis  of  the 
host.  By  injecting  into  the  guinea-pig  separate  triturations  of  the 
salivary  glands  and  aUmentary  sac  of  the  infected  tick,  the  presence 
of  the  virus  in  these  tissues  was  shown  in  1907.  It  seems  that  the 
tick  must  undergo  a  generalized  invasion  by  the  virulent  organisms, 
from  the  intestine  as  a  point  of  departure,  with  their  eventual 
localization  in  the  salivary  glands.  The  hereditary  transmission 
of  the  disease,  involving  the  presence  of  the  virus  in  the  oviduct 
and  germ  cells,  is  a  further  indication  of  this  generalized  infection. 
That  a  toxic  secretion  is  injected  at  the  time  of  feeding  is  indicated 
by  the  intense  local  reaction  which  follows  the  bite  of  even  the 
normal  tick.  There  is,  indeed,  no  difference  between  the  bite  of 
the  normal  and  the  infected  tick.  The  spotted  fever  virus  does 
not  produce  a  local  cutaneous  or  subcutaneous  reaction. 

Inheritance  in  the  tick.— In  the  summer  of  1907  three  additional 
points  were  determined  regarding  the  relationship  of  the  virus  to 
the  tick,  namely,  the  occurrence  of  hereditary  transmission,  the 

•  The  problem  of  time  relationship  is  now  the  subject  of  further  study  by  Mr.  J.  J.  Moore. 


384  Contributions  to  Medical  Science 

possibility  of  "stage-to-stage  infection,"  and  the  existence  of  natu- 
rally infected  ticks.  The  first  point  was  demonstrated  by  permit- 
ting the  offspring  of  infected  females  to  feed  on  normal  guinea-pigs, 
and  the  development  of  spotted  fever  in  the  latter  signified  the 
transmission  of  the  disease  from  the  female  to  the  larvae  through 
the  medium  of  the  germ  cell.  The  frequency  with  which  hereditary 
transmission  occurs  has  an  important  bearing  on  the  natural 
history  of  the  disease,  particularly  on  the  means  by  which  it  is 
maintained  from  year  to  year,  and  for  more  accurate  information 
in  this  regard,  the  subject  was  again  taken  up  in  the  winter  of  1907-8 
with  the  assistance  of  Dr.  Maria  B.  Maver.  In  the  neighborhood 
of  thirty  females  were  infected  and  the  virulence  of  each  was  proved 
by  means  of  a  feed  on  a  normal  guinea-pig,  after  which  they  were 
put  aside  for  the  laying  of  eggs  and  the  hatching  of  the  latter. 
Broods  were  obtained  from  only  eighteen  of  this  number,  and  of 
the  eighteen  only  nine  proved  capable  of  infecting  the  normal 
guinea-pig,  giving  us  50  per  cent  of  hereditary  transmission.  This 
percentage  is  greater  than  that  obtained  in  either  earlier  or  later 
experiments,  and  it  seems  to  be  a  safe  conclusion  that  under  natural 
conditions  hereditary  transmission  does  not  take  place  from  more 
than  50  per  cent  of  the  infected  females. 

The  practical  significance  of  this  result  is  seen  when  one  attempts 
to  answer  the  query:  How  is  spotted  fever  maintained  in  nature? 
The  occurrence  of  hereditary  transmission  explains  perfectly  how  the 
disease  is  kept  alive  from  one  spring  to  the  next,  but  does  not  explain 
its  permanent  maintenance,  since  not  more  than  50  per  cent  of  the 
infected  females  transmit  the  disease  to  their  young.  Assuming 
that  the  total  number  of  ticks  in  an  infected  region  remains  the  same 
over  a  period  of  years,  and  this  condition  appears  to  be  approxi- 
mated in  the  Bitter  Root  Valley,  a  50  per  cent  reduction  in  the 
number  of  hereditary  transmissions  for  each  succeeding  year  would, 
in  the  course  of  time,  spell  the  extermination  of  the  disease  among 
the  ticks.  There  is  no  evidence,  however,  to  show  that  spotted 
fever  is  decreasing.  It  would  seem  that  its  very  existence  at  this 
time  signifies  a  prolonged  previous  history,  and  that  there  exists 
in  nature  a  mechanism  for  the  annual  replenishment  of  the  virus 
among  the  ticks,  to  offset  the  reduction  (50  per  cent  or  greater) 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    385 

which  takes  place  every  year.  I  shall  have  occasion  to  speak  of 
this  further  when  considering  the  relationship  of  native  animals  to 
spotted  fever  and  the  source  of  infection  for  the  ticks. 

^' Stage-to-stage  infection.^' — Still  another  phase  of  the  relation- 
ship of  the  virus  of  spotted  fever  to  the  tick  would  seem  to  be  of 
importance  for  the  maintenance  of  the  disease;  this  relates  to  the 
occurrence  of  "stage-to-stage  infection."  In  the  summer  of  1907 
larvae  from  uninfected  females  were  permitted  to  feed  on  guinea- 
pigs  which  had  been  inoculated  with  spotted  fever.  After  they  had 
moulted  into  the  next  active  stage  (nymphal)  they  were  placed  with 
normal  guinea-pigs ;  the  development  of  spotted  fever  in  the  latter, 
after  being  bitten  by  the  nymphs,  proved  that  the  organisms,  when 
taken  in  by  the  larvae,  retain  their  life  and  virulence  until  the 
succeeding  active  stage  is  reached.  In  a  similar  way  it  was  demon- 
strated that  the  nymph  which  feeds  on  diseased  blood  is  still  able 
to  transmit  spotted  fever  when  it  reaches  the  adult  stage,  some 
weeks  later. 

"Stage-to-stage  infection"  would  be  of  little  significance  for 
the  maintenance  and  spread  of  spotted  fever  were  it  not  for  the 
habit  which  Dermacentor  venustus  and  D.  modestus  possess,  of 
dropping  from  the  host  for  the  process  of  moulting.*  To  assume  a 
case,  larvae  which  have  inherited  their  spotted  fever  may  infect 
a  ground  squirrel;  dropping  from  this  animal  in  order  to  moult, 
certain  of  them,  after  reaching  the  nymphal  stage,  may  reach  a 
hitherto  uninfected  ground  squirrel,  or  a  rock  squirrel,  and  convey 
the  disease  to  the  latter;  and  this  event  may  again  be  repeated 
after  the  adult  stage  is  reached.  Thus  it  is  possible  for  a  single 
tick  to  infect  two  or  three  different  animals  in  the  different  stages 
of  its  development.  The  habit  of  dropping  also  affords  opportunity 
for  extension  of  the  disease  among  the  ticks.  Non- virulent  larvae 
and  nymphs,  feeding  on  a  susceptible  animal  in  company  with 
infected  ticks,  may  well  derive  the  disease  from  the  animal  after  its 
infection  is  established,  and  they  in  turn  after  moulting  and  seeking 
further  hosts  may  cause  additional  extension  in  the  same  way. 

•  This  is  the  custom  of  most  species,  but  some,  as  Margaropus  annulatus,  which  carries  Texas  fever, 
remain  on  the  host  for  both  moults.  The  latter,  after  reaching  a  host  as  larvae,  remain  with  it  until  the  adult 
stage  is  reached,  and,  indeed,  until  the  female  is  ready  to  drop  and  lay  her  eggs.  Hence,  even  if  stage-to- 
stage  infection  occurs  in  this  instance,  it  can  have  no  great  significance  for  the  extension  of  Texas  fever. 


386  Contributions  to  Medical  Science 

Hence,  it  is  manifest  that  the  faciHties  for  extension  are  much 
greater,  in  view  of  the  habit  of  "dropping,"  than  they  would  be 
if  the  tick  remained  on  its  original  host  during  its  entire  develop- 
ment. 

From  what  has  been  said  it  may  be  inferred  that  the  ticks  in 
question  have  a  multiplicity  of  hosts.  They  are  known  to  occur 
naturally  on  at  least  seventeen  of  the  domestic  and  wild  animals 
of  the  Rocky  Mountains  (horse,  cow,  dog,  swine,  sheep,  elk,  deer, 
mountain  goat  and  sheep,  bear,  ground  squirrel,  pine  squirrel, 
rock  squirrel,  ground  hog,  chipmunk,  mountain  rat,  and  the  so- 
called  rock  rabbit),  and  experimentally  they  feed  readily  on  the 
guinea-pig,  rabbit,  and  monkey. 

Naturally  infected  ticks. — As  proof  of  the  etiological  relationship 
of  the  tick  to  spotted  fever,  I  have  cited  the  experiments  of  McCalla 
and  Brereton,  in  transmitting  the  disease  from  man  to  man,  and 
m}r  own  work  demonstrating  the  carrying  power  of  the  tick,  and 
the  intimate  relation  of  the  virus  to  the  tick,  as  illustrated  by  heredi- 
tary transmission  and  stage-to-stage  infection.  To  these  may  be 
added  the  almost  unfailing  history  of  recent  tick  bites  in  the  cases 
of  human  infection.*  With  all  these  data,  however,  it  may  still 
be  argued,  with  some  show  of  reason,  that  these  conditions  are 
accidental,  that  the  mere  fact  that  the  tick  has  the  carrying  power 
does  not  constitute  proof  that  man  acquires  his  infection  from  the 
tick.  Furthermore,  to  remove  an  infected  tick  from  a  human 
patient  and  prove  its  virulence  does  not  improve  the  character  of 
the  proof,  since  the  critic  may  say  that  the  tick  derived  its  infec- 
tion from  the  patient,  rather  than  the  converse. 

Appreciating  both  the  pertinence  and  the  improbability  of  this 
conception,  it  seemed  that  the  situation  demanded  the  discovery 
of  naturally  infected  ticks.  This  was  undertaken  successfully  in 
1907.  Several  hundred  ticks  from  an  "infected  district"  were 
allowed  to  feed  on  guinea-pigs  in  groups  of  from  thirty  to  fifty.  One 
of  these  groups  produced  spotted  fever  in  its  guinea-pig,  and  the 

*  As  stated  in  reports  to  the  state  of  Montana,  the  failure  to  find  a  tick  on  the  patient,  or  evidence 
of  a  recent  tick  wound,  provides  no  evidence  of  the  absence  of  a  tick  bite.  A  few  hours'  feed  only  is  required 
for  infection,  and  a  tick  may  feed  and  escape  or  be  brushed  off  accidentally  and  the  individual  himself 
have  no  knowledge  of  the  bite.  Tick  wounds  situated  on  the  scalp  or  perineum  may  well  escape  very  care- 
ful search. 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    387 

diagnosis  was  verified  by  repeated  passage  and  immunity  tests. 
During  the  past  spring  it  seemed  desirable  to  repeat  this  observa- 
tion. Ticks  were  collected  from  Lo  Lo  Valley,  which  is  tributary 
to  the  Bitter  Root  Valley,  and  sent  to  Chicago,  where  Dr.  Maver 
conducted  the  tests,  placing  the  ticks  with  guinea-pigs  in  groups 
as  in  the  previous  experiments.  In  this  instance  infected  ticks  were 
found  in  two  of  the  groups,  and  a  strain  of  spotted  fever  derived  in 
this  way  is  still  being  carried  on  by  the  method  of  passage  in  the 
guinea-pig. 

With  the  discovery  of  naturally  infected  ticks,  it  would  seem 
that  the  requirements  for  proof  of  the  relationship  of  the  tick  to 
the  infection  of  man  have  been  met  as  fully  as  it  is  possible  to 
meet  them. 

Seasonal  relations. — As  I  have  stated  elsewhere,  the  peculiar 
limitation  of  spotted  fever  to  the  months  of  spring  depends  on  two 
facts  in  relation  to  the  development  and  habits  of  the  ticks  involved: 
First,  that  the  cycle  of  development  is  annual,  and  second,  that 
only  the  adult  ticks  are  found  as  parasites  of  man*  and  adult  ticks 
are  to  be  found  only  in  the  spring,  or  roughly  so.  One  may,  indeed, 
find  occasional  adults  in  February  or  the  latter  part  of  January,  or, 
on  the  other  hand,  as  late  as  August  or  September.  High  in  the 
mountains  they  are  found  later  in  the  summer  than  in  the  lower  val- 
leys. Presumably,  this  is  on  account  of  the  lower  temperature  in  the 
former  localities,  which  favors  both  a  late  development,  and  a  prolon- 
gation of  life  after  the  adult  stage  is  once  reached.  Such  observations 
are  exceptional,  however,  and  it  remains  true  that  the  appear- 
ance and  activity  of  the  adult  begins  some  time  during  the  latter 
part  of  March  or  the  first  part  of  April,  the  exact  time  depending 
on  the  occurrence  of  warm  days,  and  that  they  are  not  commonly 
seen  after  the  middle  of  June.  We  may  be  quite  sure  that  the 
temperature  of  the  region  is  the  most  potent  factor  in  determining 
this  rather  sharp  annual  cycle.  From  experience  gained  in  labora- 
tory cultivation,  and  by  observations  on  the  stock  and  wild  animals, 
we  may  fix  the  egg-laying  period  in  nature  in  the  latter  part  of 
the  spring  and  the  early  part  of  the  summer.     During  the  summer 

*  Only  in  two  instances  have  I  learned  of  larvae  and  nymphs  feeding  on  man;  children  were  concerned 
in  both  instances. 


388  Contributions  to  Medical  Science 

larvae  and  nymphs  are  found  on  the  small  wild  animals  in  large 
numbers,  and  it  is  probable  that  many  of  the  nymphs  have  had 
their  feed  and  dropped  from  their  hosts  before  the  onset  of  cold 
weather.  In  several  instances,  however,  large  numbers  of  feeding 
nymphs  have  been  obtained  from  horses  in  December  and  January, 
hence  it  is  probable  that  the  nymphal  development  is  in  progress 
during  the  most  of  the  winter.  Those  which  drop  from  the  hosts 
late  in  the  autumn  probably  spend  the  entire  winter  in  the  resting 
or  moulting  stage,  which  they  complete  on  the  advent  of  warmer 
weather.  Little  progress  is  made  in  moulting  during  the  winter. 
Enlarged  nymphs  have  been  kept  in  the  laboratory  at  a  tempera- 
ture of  about  10°  C.  for  nine  months  without  moulting,  but  when 
removed  to  the  temperature  of  the  room,  the  process  would  go  on 
to  completion  in  about  the  usual  time.  The  enlarged  nymphs 
which  drop  from  their  hosts  at  any  time  during  the  winter  must 
wait  for  the  warm  weather  of  spring  before  they  can  cast  their 
skins  and  appear  as  adults. 

Source  of  virus  for  the  tick. — The  observations  in  relation  to 
hereditary  transmission  in  the  tick  indicate,  as  stated  previously, 
that  it  is  necessary  to  assume  some  extraneous  source  of  infection 
for  the  tick  in  order  to  explain  the  maintenance  of  the  disease. 

At  the  outset  it  would  be  natural  to  inquire  whether  man  can 
play  the  same  role  in  the  maintenance  of  spotted  fever  as  he  does 
in  that  of  malaria  and  yellow  fever,  and  as  he  possibly  does  also  in 
the  maintenance  of  human  tick  fever  of  South  Africa.  Without 
going  into  a  full  discussion  of  all  the  conditions  which  would  seem 
to  render  this  impossible,  the  essential  reasons  for  this  conclusion 
may  be  given  as  follows: 

Ticks  which  are  naturally  infected  have  no  relation  to  the  habita- 
tion of  man;  they  are  wood- ticks,  not  house  pests,  and  are  not 
found  in  the  houses  except  as  they  are  brought  in  accidentally  on 
the  clothing.  The  conditions  are  very  different  in  the  relation  of 
Ornithodorus  moubata  to  South  African  tick,  or  recurrent,  fever. 
The  latter  have  the  habits  of  the  bedbug  to  a  large  degree,  secreting 
themselves  in  the  house  during  the  day  and  coming  out  at  night 
to  feed  on  the  inmates.  In  this  case  man  may  well  be  the  common 
or  only  source  for  the  fresh  infection  of  the  carriers.     For  man  to 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    389 

be  concerned  in  the  maintenance  of  spotted  fever  it  would  be 
necessary  to  assume  that  ticks  which  were  hitherto  normal  must 
feed  on  him  during  the  course  of  his  illness,  and  that  the  females 
must  either  mature  on  him  directly  in  preparation  for  the  laying  of 
eggs,  or,  after  leaving  him,  must  subsequently  find  some  other  host 
on  which  it  may  mature.  One  who  has  seen  the  enormous  size 
which  the  matured  female  attains,  and  who  knows  that  a  continu- 
ous feed  of  not  less  than  a  week  or  ten  days  is  required  for  this 
degree  of  development,  can  hardly  conceive  of  its  occurrence  on  a 
patient  who  has  been  stripped  of  clothing  and  is  confined  in  bed. 
Spotted  fever  patients  are  invariably  looked  over  carefully  for  ticks, 
and  it  is  a  safe  conclusion  that  those  which  are  found  are  destroyed 
with  little  ceremony.  Regarding  the  possibihty  that  a  tick  after 
once  feeding  on  a  patient  may  escape  and  subsequently  find  another 
host  on  which  it  may  mature,  this  must  be  admitted  as  a  rare 
possibility,  or  better  as  an  extreme  improbability.  The  tick 
travels  slowly,  covers  very  short  distances,  and  does  not  readily 
find  a  second  host.  On  account  of  the  virtual  impossibility  of  a 
female  maturing  on  man,  we  should  have  to  assume  that  the  second 
host  in  this  case  would  be  an  animal  rather  than  another  human. 
The  dog,  and  perhaps  the  cat  (?),  exist  as  the  only  reasonable 
possibilities  in  the  way  of  such  a  second  host,  and  they  alone  render 
the  occurrence  under  consideration  a  rare  possibility.  Given 
even  the  matured  and  infected  female,  the  likehhood  of  the  off- 
spring being  infected  is  not  more  than  50  per  cent,  and,  further- 
more, the  chances  which  operate  against  the  development  of  the 
progeny  of  any  one  female  to  the  succeeding  adult  stage  are  very 
heavy.  Hence,  for  these  various  reasons  the  process  mentioned 
virtually  drops  out  of  consideration  as  a  means  for  the  maintenance 
of  spotted  fever. 

The  study  of  small  wild  animals. — Holding  to  the  correctness  of 
the  conclusion  just  stated,  it  seemed  necessary,  in  a  search  for  the 
source  of  infection  for  the  tick,  to  turn  to  its  more  general  food 
supply,  namely,  to  one  or  more  of  the  domestic  or  wild  animals 
on  which  the  arachnid  feeds  more  or  less  extensively.  The  question 
may  be  raised  as  to  whether  it  is  justifiable  to  exclude  the  vege- 
table kingdom  from  an  investigation  of  this  character.     In  answer 


390  Contributions  to  Medical  Science 

one  can  only  say  that  the  probabilities  of  the  case  seemed  to  justify 
such  an  exclusion.  It  is  the  general  and  firm  belief  among  ento- 
mologists that  such  ticks  live  exclusively  on  the  blood  of  animals, 
and  that  the  anatomy  of  their  mouth  parts  permits  of  no  other 
type  or  mechanism  of  nourishment.  I  have  undertaken  no  work 
to  determine  whether  or  not  the  life  of  ticks  may  be  prolonged  by 
keeping  them  in  the  presence  of  various  types  of  living,  dead,  or 
decaying  vegetables,  or,  for  that  matter,  with  dead  or  decaying 
animal  tissues.  In  a  fairly  extensive  experience,  however,  they 
have  never  been  observed  in  the  position  of  feeding  on  such  vege- 
tables as  are  kept  with  the  animals  in  the  tick-proof  cages,  namely, 
carrots,  cabbage,  dandelions,  and  clover.  Concerning  the  possi- 
bility that  they  may  feed  on  dead  or  decaying  animal  matter,  it 
can  only  be  said  that  ticks,  as  a  rule,  make  haste  to  leave  the  body 
of  an  animal  on  which  they  have  been  feeding  after  the  death  of 
the  latter.  As  a  determining  influence,  moreover,  we  had  before 
us  the  demonstrated  fact  that  the  tick  readily  acquires  spotted 
fever  by  feeding  on  the  blood  of  an  infected  animal,  and  it  seemed 
rational  to  assume  that  it  would  acquire  the  disease  in  a  similar 
manner  under  natural  conditions. 

Hence,  a  campaign  was  outhned,  which  had  as  its  object  a  study 
of  the  various  animals  which  serve  as  natural  hosts  for  the  tick — 
a  campaign  which  has  not  yet  been  brought  to  a  conclusion,  but 
which  has  yielded  results  so  suggestive  in  their  character  as  to  fur- 
nish an  ample  reward  for  the  labor  and  difiiculties  involved. 

Certain  types  of  animals  were  eliminated  from  the  study  for 
what  appeared  to  be  justifiable  reasons.  Because  of  the  limita- 
tion of  spotted  fever  to  the  Bitter  Root  Valley,  in  so  far  as  western 
Montana  is  concerned,  it  was  decided  to  postpone  the  study  of 
animals  which  wander  widely.  Hence  deer,  elk,  bear,  birds,  and 
animals  (mountain  goat  and  sheep)  which  continuously  remain 
remote  from  human  habitation  were  omitted  from  the  list,  at  least 
for  the  time  being.  Also,  because  of  the  demonstrated  resistance 
of  the  horse  (relative  resistance),  ox,  sheep,  and  the  fowl,  the  domes- 
ticated animals  were  not  subjected  to  further  study  for  the  purpose 
under  discussion. 

Just  one  definite  clue  served  to  indicate  the  type  of  animal 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    391 

which,  it  appeared,  should  be  selected  for  the  first  experimentation. 
This  concerns  the  segregation  of  spotted  fever  not  only  in  the  Bitter 
Root  Valley  as  contrasted  with  adjacent  districts  in  which  the  ticks 
and  animal  life  are  identical,  but  also  its  localization  to  the  west 
slope  of  the  valley,  and  even  to  particular  foci  on  the  west  slope. 
On  the  basis  of  this  consideration  it  would  seem  that  an  animal 
to  be  concerned  in  maintenance  must  be  one  which  has  a  tendency 
to  a  more  or  less  permanent  abode,  one  which  does  not  wander 
widely  from  its  habitation  from  year  to  year,  hence  one  which 
would  not  be  likely  to  disseminate  infected  ticks  over  a  large 
area. 

The  ground  squirrel  (a  spermophile) ,  ground  hog  or  woodchuck, 
the  rock  squirrel,  the  chipmunk,  the  pine  squirrel,  rock  rabbit, 
mountain  rat,  and  perhaps  the  three  species  of  rabbit  found  in  the 
district  (cotton-tail,  snow-shoe,  and  jack  rabbit)  were  considered 
as  meeting  this  requirement  more  closely  than  other  species. 

Briefly,  the  plan  was:  first,  to  determine  the  susceptibiHty  of 
these  species  as  it  might  be  indicated  by  the  injection  of  virulent 
blood;  second,  with  those  which  were  found  to  be  susceptible  by 
the  method  of  injection,  to  attempt  infection  by  means  of  the  bites 
of  ticks  of  known  virulence;  third,  to  perform  "tick  cycle"  experi- 
ments, by  which  is  meant  the  production  of  the  disease  by  means  of 
the  tick,  while  at  the  same  time  permitting  normal  ticks  to  acquire 
the  disease  by  feeding  on  the  animal  during  the  course  of  fever; 
fourth,  and  last,  to  attempt  to  discover  individual  animals  in  nature, 
which  were  either  suffering  from  spotted  fever  at  the  time  of  obser- 
vation, or  which  had  survived  an  infection  at  some  previous  time. 
The  last  point  was  to  be  determined  by  means  of  immunity  tests, 
provided,  of  course,  that  the  previous  experimentation  should 
show  that  one  attack  in  the  various  species  confers  lasting  im- 
munity.    It  has  not  yet  been  attacked  experimentally. 

It  is  to  be  understood  that  in  the  study  of  the  first  three  points, 
the  animals  used  in  the  experiments  were  taken  from  localities  in 
which  spotted  fever  had  not  been  known  to  occur. 

The  first  experiments  with  the  ground  squirrel  were  perplexing. 
It  had  been  anticipated  that  a  rise  in  the  temperature  of  this  animal 
would  serve  as  a  guide  to  the  occurrence  of  infection,  as  it  does  in 


392  Contributions  to  Medical  Science 

the  case  of  the  guinea-pig,  rabbit,  and  monkey.  The  results, 
however,  were  very  inconstant  in  this  regard.  In  four  out  of 
thirteen  animals  inoculated,  four  had  absolutely  no  fever.  The 
shortest  febrile  period  observed  was  one  day,  and  it  persisted  no 
longer  than  four  consecutive  days  in  any  of  them;  in  four  animals 
it  lasted  for  three  days,  and  in  another  for  two.  "The  incubation 
period  varied  from  one  to  five  days.  It  was  five  days  in  one  animal, 
four  days  in  two,  three  days  in  four,  two  days  in  one,  and  one  day 
in  one  animal.  During  the  incubation  period  the  temperature 
varied  between  ioo°  and  103°,  being  for  the  most  part  below  102 . 5°. 
One  observation  was  as  low  as  97 . 8°.  After  the  cessation  of  fever 
the  temperature  returned  to  about  what  it  was  during  the  incubation 
period."'  But  one  of  the  animals  inoculated  died,  and  at  autopsy 
the  only  discoverable  change  was  a  pronounced  enlargement  of  the 
spleen,  as  compared  with  controls  which  were  killed  for  the  purpose 
of  comparison.  From  this  animal,  and  also  from  one  other  which 
exhibited  distinct  fever,  transfers  of  blood  were  made  into  the 
guinea-pig,  and  in  both  instances  the  latter  developed  typical 
attacks  of  spotted  fever,  as  shown  by  the  fever,  hemorrhages  into, 
and  gangrene  of,  the  scrotum,  and  the  changes  in  the  spleen  and 
lymph  glands  seen  at  autopsy. 

The  series  showed  the  possibility  of  infecting  the  ground  squirrel 
with  spotted  fever,  but  it  seemed  necessary  to  conclude  either  that 
the  susceptibility  of  the  different  individuals  is  not  uniform,  or 
that  the  temperature  which  follows  injection  cannot  be  taken  as  an 
index  of  infection. 

With  the  hope  of  obtaining  information  regarding  the  last  point, 
"the  temperatures  of  a  number  of  normal  gophers  were  taken  for 
several  consecutive  days  in  order  to  learn  its  natural  variations. 
It  was  found  to  be  very  inconstant  and  the  irregularities  were  not 
always  to  be  accounted  for.  When  the  weather  was  cool,  as  in  the 
mornings,  it  was  common  to  observe  a  low  temperature,  and  for 
several  days  it  might  remain  in  the  vicinity  of  97°  or  98°  F.  At 
other  times  it  was  low  when  the  outside  temperature  was  too  high 
to  account  for  the  condition.  That  of  the  normal  animal  rarely 
exceeded  102.5°  or  103°  F.,  and  it  was  concluded  that  this  would 
represent  approximately  its  temperature  when  in  a  state  of  normal 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    393 

activity,  and  with  metabolism  at  par.  The  fact  that  the  gopher 
is  a  hibernating  and  a  semi-cold-blooded  animal,  no  doubt  is 
responsible  for  many  of  these  variations.  When  in  actual  hiberna- 
tion the  temperature  in  three  instances  was  so  low  that  it  did  not 
register  on  the  clinical  thermometer.  It  has  been  observed,  also, 
that  there  are  individual  variations  in  the  tendency  to  go  into  the 
somnolent  state,  that  even  at  the  warm  temperature  of  the  labora- 
tory one  may  be  so  somnolent  that  he  can  be  taken  out  and  handled 
without  awakening,  while  others  will  be  quite  active.  It  seemed 
more  probable,  therefore,  that  the  temperature  of  the  gopher  is 
not  a  good  index  of  his  infection  rather  than  that  30  per  cent  of 
the  species  should  be  naturally  resistant  to  the  disease." 

During  the  following  spring  and  summer  (1908)  this  was  proved 
to  be  true.  Of  a  series  of  squirrels  which  received  quantities  of 
virus  from  the  guinea-pig  varying  from  0.02  c.c.  to  3.0  c.c,  and 
which  showed  the  greatest  irregularities  in  temperature  following 
the  injections,  every  one  acquired  spotted  fever,  the  diagnosis 
being  established  by  the  injection  of  blood  from  each  of  the  animals, 
at  the  proper  time,  into  normal  guinea-pigs. 

It  may  be  mentioned  as  a  point  of  incidental  interest  that  a 
relative  fever  developed  in  a  large  percentage  of  the  ground  squirrels. 
If  the  temperature  of  a  particular  animal  had  been  found  in  the 
neighborhood  of  98°  or  99°  for  several  days,  it  would  commonly 
rise  to  a  height  of  101°  to  103°  during  the  customary  febrile  period, 
the  latter  being  scarcely  above  that  of  the  normal  active  animal. 

The  experience  taught  a  new  point  in  technic,  namely,  that 
for  the  positive  recognition  of  spotted  fever  in  the  ground  squirrel 
it  is  necessary  to  carry  the  inoculation  back  again  from  the  latter 
to  the  normal  guinea-pig.  Eventually  this  step  was  adopted  as  a 
uniform  procedure  in  the  work  with  other  small  wild  animals. 

Following  this  plan  of  experimentation,  the  ground  squirrel, 
ground  hog,  rock  squirrel,  chipmunk,  and  mountain  rat  were  found 
to  be  susceptible  to  the  inoculation  of  the  virus  of  spotted  fever, 
as  represented  in  the  blood  of  the  infected  guinea-pig  on  the  third 
day  of  fever.  The  first  four  species  were  also  found  to  be  suscep- 
tible to  the  bite  of  single  virulent  adult  ticks,  the  infectivity  of 
which  had  been  proved  previously  on  normal  guinea-pigs.     It  was 


394  Contributions  to  Medical  Science 

not  possible  at  the  time  to  study  the  susceptibiUty  of  the  mountain 
rat  to  the  bite  of  the  infected  tick. 

It  is  of  theoretical  and  practical  importance  to  know  whether 
spotted  fever  is  an  acute  self-Kmited  infection  in  the  animals  under 
consideration,  or  whether  it  may  exist  in  a  chronic  form.  If  it 
were  sufficiently  chronic,  lasting  for  a  year  or  more,  as  in  the  case 
of  certain  protozoan  diseases,  maintenance  could  be  accomplished 
without  the  aid  of  inheritance  in  the  tick.  Inasmuch  as  the  latter 
does  occur  the  only  evident  influence  of  chronicity  in  the  animal 
would  be  a  more  massive  infection  of  the  ticks  of  the  locaHty. 
Investigations  have  shown,  however,  that  the  number  of  infected 
ticks,  in  comparison  with  the  whole,  is  very  small — so  small  indeed 
that  this  fact  alone  would  seem  to  be  a  strong  argument  against 
chronicity  in  the  four-footed  hosts. 

Two  general  arguments  also  speak  for  acuteness  in  the  small 
wild  hosts:  first,  that  in  the  other  animals  which  have  been  studied 
carefully,  and  in  man,  the  disease  is  acute  and  self-limited;  second, 
that  after  recovery  protracted  (probably  permanent)  immunity 
is  estabUshed  against  reinfection,  and  the  serums  of  such  animals 
possess  protective  antibodies  in  considerable  concentration — much 
more  than  is  found  in  any  known  chronic  disease. 

In  only  two  of  the  wild  animals  has  the  subject  been  studied 
specifically,  the  ground  squirrel  and  the  rock  squirrel.  After  they 
have  recovered,  the  blood  is  free  from  virus,  as  shown  by  transfers 
into  the  guinea-pig,  and  they  resist  attempts  at  reinoculation. 
One-half  c.c.  of  the  blood  of  the  immune  ground  squirrels  was  found 
to  protect  the  guinea-pig  against  approximately  i,ooo  pathogenic 
doses  of  virus,  the  blood  of  the  normal  squirrel  having  no  protec- 
tive effect.  The  possible  presence  of  the  virus  in  some  of  the 
internal  organs  of  these  animals  after  recovery  has  not  been  investi- 
gated. The  concentration  of  antibodies  in  the  blood  would  seem 
to  preclude  this.  It  is  not  found  in  the  spleen  of  the  guinea-pig 
and  monkey  after  recovery. 

It  is  manifest  from  previous  statements  that  spotted  fever  in 
the  tick  is  very  chronic.  The  relation  of  the  disease  to  the  tick, 
indeed,  does  not  differ  from  that  of  various  microparasites  to  their 
insect  carriers  in  this  respect. 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    395 

^^ Tick-cycle'^  experiments. — The  "tick-cycle"  experiments  repre- 
sented an  attempt  to  duplicate  what  was  assumed  to  be  a  step  in 
the  maintenance  of  the  disease  in  nature.  Individual  ticks,  rather 
than  groups  of  ticks,  were  dealt  with,  and  the  following  steps  were 
involved : 

First,  the  individual  testing  of  virulent  ticks  on  separate  guinea- 
pigs  in  order  to  prove  their  infectivity  before  beginning  the 
experiment;  second,  the  testing  of  the  supposedly  normal  ticks  on 
guinea-pigs  to  insure  their  freedom  from  the  virus ;  third,  the  attach- 
ment of  a  single  infected  tick  to  the  ear  or  head  of  the  small  wild 
animal,  permitting  the  former  to  feed  for  a  period  which  was  known 
to  be  sufficient  for  the  production  of  the  disease;  fourth,  after  the 
lapse  of  an  interval  corresponding  to  the  established  incubation 
period,  to  draw  blood  from  the  heart  of  the  experiment  animal,  and 
inject  it  into  the  guinea-pig  for  the  purpose  of  determining  the  ex- 
istence or  non-existence  of  spotted  fever  in  the  wild  animal;  fifth, 
the  attachment  of  one  or  more  normal  ticks  to  the  latter  as  soon  as 
the  incubation  period  has  passed;  and  sixth,  the  testing  of  the  latter 
on  normal  guinea-pigs  in  order  to  determine  whether  or  not  they 
had  acquired  the  virus  during  their  feed. 

There  were  many  failures  and  embarrassments  due  to  the  acci- 
dental death  of  valuable  ticks,  or  of  animals,  or  to  failure  to  do  the 
right  thing  at  the  right  time  in  all  cases;  but  eventually  positive 
"tick-cycle"  experiments  were  obtained  with  the  ground  squirrel, 
ground  hog,  and  rock  squirrel.  With  the  delicate  Httle  chipmunk 
the  cycles  were  broken  in  character.  Several  times  this  animal  was 
infected  by  the  bite  of  virulent  ticks,  and  we  were  successful  in 
transferring  the  disease  from  the  chipmunk  to  the  tick,  following 
the  inoculation  of  the  former  by  means  of  the  syringe.  In  the 
"cycle"  experiments,  however,  the  intermediate  step  of  bleeding 
from  the  heart  proved  fatal  in  nearly  all  cases,  which,  of  course, 
prevented  the  completion  of  the  experiment.  Nevertheless,  the 
broken  character  of  the  experiments  with  the  chipmunk  does  not 
materially  impair  their  significance. 

These  experiments  would  have  little  justification  were  it  not  for 
the  fact  that  the  animals  mentioned  are  utilized  as  hosts  by  the  tick 
at  one  stage  or  another  of  its  development.     Particularly  on  the 


396  Contributions  to  Medical  Science 

ground  squirrel,  rock  squirrel,  and  pine  squirrel  (the  last  not  yet 
studied),  larvae  and  nymphs  have  been  found  in  great  abundance, 
and  on  the  other  animals  mentioned,  to  a  less  extent.  That  adult 
ticks  are  found  less  commonly,  indeed  rarely  in  my  experience, 
on  these  animals  than  the  earher  stages,  in  no  way  invalidates  the 
experiments,  which  were  performed  with  the  adults,  since  an  abun- 
dance of  experience  shows  that  the  larvae  and  nymphs  do  not  differ 
from  the  adults  in  their  ability  to  acquire  and  transmit  spotted 
fever.  We  may  only  bear  in  mind  the  possibility  that  more  than 
one  larva  may  be  required  for  the  production  of  the  disease,* 
whereas  a  single  nymph  or  adult  is  sufficient. 

Maintenance. — In  accordance  with  the  results  and  deductions 
which  have  been  outlined,  it  is  conceived  that  spotted  fever  is 
maintained  as  follows:  A  certain  percentage  of  the  female  ticks 
which  have  acquired  the  disease  as  a  consequence  of  feeding  on 
animals,  the  latter  having  been  infected  by  other  ticks,  transmit 
the  disease  to  their  offspring  through  the  egg.  The  new  genera- 
tion during  the  process  of  feeding  transfer  the  virus  to  certain  of 
the  susceptible  small  wild  animals  (ground  squirrels,  rock  squirrels, 
chipmunks,  ground  hogs,  and  perhaps  others),  and  this  may  take 
place  during  either  the  larval,  nymphal,  or  adult  stage,  hence  at 
various  times  of  the  year.  During  the  infection  of  the  wild  animal 
it  is  required  that  hitherto  normal  ticks,  either  as  larvae,  nymphs, 
or  adults,  acquire  the  disease  by  feeding  simultaneously  with,  or 
shortly  after,  the  feeding  of  the  infected  ticks.  Regardless  of  the 
tick's  stage  of  development  at  the  time  it  acquired  the  disease,  the 
virus  is  retained  into  the  adult  period,  and  in  certain  of  the  females 
reaches  the  germ  cells  and  again  appears  in  the  next  generation. 
The  infection  of  man  is  an  unessential  incident  for  maintenance, 
and  depends  on  the  occasional  and  accidental  bite  of  the  infected 
adult  tick. 

I  shall  not  discuss  the  bearing  which  these  results  have  on  the 
control  and  the  eradication  of  the  disease  further  than  to  say  that 
this  work  naturally  should  take  two  directions,  one  having  as  its 
object  the  destruction  of  the  ticks  in  proximity  to  human  habitation, 

*  In  testing  the  virulence  of  eggs,  five  is  the  smallest  number  which  so  far  has  proved  capable  of  infect- 
ing the  guinea-pig. 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    397 

following  methods  which  have  proved  successful  in  the  cam- 
paign against  Texas  fever,  and  the  other  leading  to  the  extermina- 
tion of  the  susceptible  wild  species  of  animals,  a  procedure  which 
also  has  as  a  consequence  the  curtailment  of  the  food  supply  for 
the  ticks.  Detailed  recommendations  embodying  these  points 
have  been  made  to  the  State  Board  of  Health  of  Montana. 

Microbic  etiology. — Inasmuch  as  rational  methods  for  the  control 
and  eradication  of  spotted  fever  depend  on  an  accurate  acquaint- 
ance with  the  means  by  which  man  is  infected,  and  the  agencies 
through  which  the  disease  is  maintained  in  nature,  it  is  fortunate 
that  these  questions  could  be  investigated  without  any  knowledge 
of  the  micro-organism  which  causes  the  disease.  Yet  so  much 
theoretical,  and  perhaps  practical,  importance  pertains  to  the 
recognition,  and  particularly  to  the  cultivation,  of  the  causative 
agent,  that  this  part  of  the  problem  has  never  entirely  dropped  out 
of  mind.  Following  many  failures,  further  study  was  often 
deferred  until  other  lines  of  work  which  promised  solution  could 
be  disposed  of. 

With  the  knowledge  that  the  tick  is  the  agent  through  which 
man  is  infected,  one  turns  naturally,  and  first  of  all,  to  some  other 
diseases  of  known  etiology  which  are  also  carried  by  one  or  another 
species  of  tick,  at  the  same  time  keeping  in  mind  the  dangers  of 
generalizations  and  analogies.  When  spotted  fever  was  first 
studied  ticks  were  known  as  the  carriers  of  piroplasmas  only.  On 
the  other  hand,  since  1903,  it  has  been  definitely  shown  that  ticks 
are  the  medium  of  conveyance  in  at  least  three  diseases  which  are 
caused  by  spirilla,  the  spirillosis  of  fowls  (Marchoux  and  Salembini), 
South  African  tick  fever  of  man  (Button  and  Todd,  and  Koch), 
and  a  disease  of  cattle  in  South  Africa  (Theiler) . 

With  these  analogies  in  mind,  and  animated  also  by  the  con- 
flicting opinions  of  Wilson  and  Chowning  on  the  one  hand,  and 
Stiles  on  the  other,  a  careful  search  has  been  carried  on  for  both 
piroplasmas  and  spirilla,  but  with  absolutely  negative  results  in 
both  cases.  If  it  is  fair  to  judge  from  the  known  infections  with 
piroplasmas  and  spirilla,  it  would  seem  that  the  recognition  of  these 
organisms  should  not  be  attended  with  great  difficulties,  particu- 
larly since  the  introduction  of  the  Romanowsky  method  of  staining, 


398  Contributions  to  Medical  Science 

with  the  various  convenient  modifications.  In  the  case  of  the 
known  piroplasmoses  the  blood  is  often  heavily  infected,  with  many 
parasites  in  a  single  field,  and  even  the  most  minute  species,  as 
Piroplasma  parvum  in  the  cattle  of  South  Africa,  are  of  such  size 
in  relation  to  the  erythrocytes  that  they  could  not  escape  detection 
in  prolonged  microscopic  studies  which  were  carried  on,  during 
all  stages  of  the  disease,  with  both  the  blood  and  the  various 
internal  organs.  Such  studies  were  entirely  negative  with  both 
fresh  and  stained  material,  and  after  many  months  of  study  in 
which  fresh  material  was  always  at  hand  the  conclusion  was 
reached  that  spotted  fever  either  is  not  caused  by  a  piroplasma 
at  all,  or  if  it  is,  that  the  organism  is  extremely  minute  (ultra- 
microscopic)  and  not  susceptible  to  microscopic  demonstration. 

There  are  also  some  general  considerations  which  suggest 
strongly  that  spotted  fever  is  not  a  piroplasmosis.  In  the  first 
place  there  is  no  massive  destruction  of  the  erythrocytes  in  either 
man  or  the  guinea-pig,  the  number  of  which  does  not  fall  below  a 
level  commonly  seen  in  various  febrile  infections.  There  is  no 
hemoglobinuria,  such  as  is  seen  in  many  piroplasmoses,  and  the 
degree  of  icterus  is  not  more  than  frequently  occurs  in  different 
fevers  of  bacterial  cause,  nor  is  the  blood  pale  and  watery.  Further, 
the  facts  that  spotted  fever  in  man  is  invariably  an  acute  self- 
limited  disease  and  that  the  serum  of  man  and  animals  after 
recovery  possesses  protective  antibodies  in  rather  high  concentra- 
tion are  not  in  harmony  with  a  piroplasmatic  etiology. 

The  results  of  the  search  for  spirilla  were  similar.  In  relapsing 
fever,  in  the  South  African  tick  fever  of  man,  and  in  the  spirilloses 
of  animals  the  number  of  organisms  in  the  circulation  is  often  very 
conspicuous.  In  spotted  fever,  on  the  other  hand,  exhaustive 
search  of  the  blood  and  tissues  in  all  stages  of  the  disease  and 
painstaking  examination  of  the  tissues  and  eggs  of  infected  ticks 
have  failed  to  reveal  the  presence  of  spirilla.  Here  again  it  must  be 
concluded  that  if  spirilla  are  present  they  are  so  small  as  to  justify 
the  assumption  that  they  would  be  ultramicroscopic  or  filterable. 

Similarly  the  possibility  of  a  leukocytic  parasite  has  always 
been  kept  in  mind,  but  such  forms  have  not  been  observed. 

From  time  to  time  various  experiments  were  performed  with 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    399 

the  hope  that  some  light  might  be  thrown  on  the  character  of  the 
organism.  The  virus  was  found  to  have  a  fairly  uniform  distribu- 
tion in  the  various  body  fluids  and  organs.  It  proved  not  to  be  an 
exclusive  parasite  of  either  the  red  or  white  cells  of  the  blood,  but 
was  found  in  approximately  the  same  concentration  in  the  lymph 
and  serum  as  in  the  full  blood.  Its  resistance  to  heat  and  light 
and  plasmolysis*  was  such  that  no  conclusion  could  be  drawn 
regarding  its  bacterial  or  protozoan  nature.  None  of  these  indirect 
methods  furnished  evidence  of  particular  value  relating  to  the  char- 
acter of  the  organism. 

A  generalization  frequently  made  in  former  years  emphasized 
the  hypothesis  that  all  diseases  which  are  transmitted  habitually 
by  insects  are  probably  due  to  protozoan  parasites,  the  assump- 
tion resting  on  facts  which  were  known  in  relation  to  many  insect- 
borne  diseases,  as  malaria,  piroplasmosis,  the  malaria  of  the  birds, 
and  more  recently,  trypanosomiasis.  Plainly,  however,  the  newer 
developments  regarding  the  relation  of  the  flea  to  plague,  and  the 
tick  to  the  relapsing  fever  of  South  Africa,  both  bacterial  diseases, 
remove  the  ground  for  the  generalization,  and  in  facing  a  new 
disease  of  this  type  today,  the  possibiUty  of  a  bacterial  rather  than 
a  protozoon  etiology  demands  unprejudiced  consideration.  If, 
indeed,  one  were  to  resort  to  generalization  in  the  case  of  spotted 
fever,  it  may  well  be  in  favor  of  a  bacterial  instead  of  a  protozoan 
parasite,  because  of  the  chronicity  and  low  grade  of  immunity  which 
pertain  to  the  known  protozoan  infections.  Even  this,  however, 
cannot  be  ventured  with  justifiable  assurance  because  of  the  num- 
ber of  diseases  whose  etiology  is  still  unknown. 

Interest  in  the  micro-organism  has  been  sustained  by  the  results 
of  filtration  experiments.  Repeatedly,  virulent  serum,  both  when 
concentrated  and  greatly  diluted,  lost  its  infective  power  after 
being  passed  through  Berkefeld  candles.  It  is,  of  course,  con- 
ceivable that  the  filter  may  have  been  more  or  less  occluded  by 
the  large  number  of  albuminous  particles  in  the  serum,  a  condition 
which  would  interfere  with  the  passage  of  the  organism  through 
the  filter.  Later,  when  dealing  with  virulent  eggs  from  the  tick, 
the  filtration  experiment  was  repeated  under  more  favorable  con- 

*  Experiments  conducted  in  association  with  Dr.  P.  G.  Heinemann. 


400  Contributions  to  Medical  Science 

ditions.  In  one  instance,  200  eggs  were  crushed  and  thoroughly 
shaken  in  5  c.c.  of  salt  solution.  The  albumin  was  so  scant  that 
even  a  faint  degree  of  opalescence  could  not  be  detected  in  the  salt 
solution.  This  was  filtered  and  the  candle  washed  with  5  additional 
c.c.  of  salt  solution.  Of  the  filtrate  a  quantity  representing  150 
eggs  was  injected  into  one  guinea-pig,  and  another,  representing 
50  eggs,  into  a  second.  Neither  of  the  animals  developed  spotted 
fever  although  5,  10,  and  15  of  the  unfiltered  eggs  produced  the 
disease  in  controls.  This  was  accepted  as  proving  finally  the  non- 
filterability  of  the  virus  through  the  ordinary,  unmodified  Berkefeld 
candle,  and  is  further  evidence  that  the  organism  is  of  such  size 
that  it  should  be  seen  with  the  microscope. 

Following  this  brief  account  of  negative  findings,  reference  may 
now  be  made  to  a  bacillus  which  has  been  found  in  association 
with  the  disease  under  interesting  circumstances.  In  the  early 
part  of  the  studies  a  diplococcus-like  body  with  an  eosin  staining 
intermediate  substance  was  encountered,  first  in  the  blood  of  an 
infected  monkey.  Further  examination  disclosed  the  presence 
of  similar  bodies  in  the  blood  of  infected  guinea-pigs,  and  later  in 
man,  and  it  came  to  be  the  common  experience  that  they  could  be 
found  in  any  preparation  of  infected  blood  during  a  search  of  not 
more  than  a  half-hour's  duration,  the  specimens  being  stained 
with  Giemsa's  solution  after  fixation  in  absolute  alcohol.  The 
morphology  varies  somewhat  regarding  the  size  and  proximity 
of  the  two  staining  bodies,  and  even  small  bacillary  forms  are 
encountered.  In  spite  of  this  constant  observation  the  conditions 
were  such  that  a  positive  interpretation  was  impossible  merely  on 
the  basis  of  the  microscopic  findings.  The  forms  are  very  minute 
and  often  appear  as  a  pair  of  granules  which  might  arise  from  a 
disintegrated  leukocyte  or  from  a  fragmented  nucleus.  As  ren- 
dering the  latter  interpretation  plausible,  disintegrating  leukocytes 
frequently  occur  in  the  blood,  particularly  of  human  patients,  and 
in  such  examples  small  globules  of  chromatin  may  sometimes  be 
found  in  proximity  to  the  injured  cell.  Further,  my  own  attempts, 
and  the  attempts  of  Mr.  P.  G.  Heinemann,  to  cultivate  an  organ- 
ism of  this  or  any  other  character  from  the  blood  of  patients  and 
animals  had  been  fruitless. 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    401 

Some  months  ago  it  was  possible  to  take  up  seriously  a  long- 
deferred  line  of  investigation,  namely,  a  study  of  the  organs  and 
eggs  of  the  tick  with  the  hope  that  the  micro-organism  might  be 
present  in  such  concentration  or  form  that  its  identification  would 
be  possible.  The  disease  being  hereditary  in  the  tick,  it  seemed 
that  the  freshly  laid  eggs  might  be  a  most  favorable  medium  for 
search  as  in  South  African  tick  fever,  one  in  which  there  would 
be  comparatively  few  opportunities  for  confusion.  The  first 
preparations  from  a  batch  of  virulent  eggs  showed  the  presence  of  an 
astounding  number  of  organisms  which,  for  the  most  part,  have  the 
form  of  minute  polar  staining  bacilli.  Frequently,  two  or  three 
are  attached  end  to  end,  and  the  staining  poles  give  the  appearance 
of  a  chain  of  four  or  six  cocci.  In  the  eggs,  as  a  rule,  the  amount 
of  intermediate  substance  interposed  between  the  poles  is  compara- 
tively large,  the  stained  masses  being  rather  widely  separated. 
Frequently,  however,  the  poles  are  more  closely  approximated, 
such  forms  resembling  closely  the  "  diplococcoid "  bodies  seen  in 
infected  blood. 

Still  other  bacilli  are  found  which  stain  solidly,  and  they  too  are 
often  in  pairs  (diplobacilli) .  In  the  latter  it  appears  that  the 
staining  substance  has  not  yet  become  segregated  into  the  poles 
of  the  organism.  Intermediate  forms  in  which  the  chromatin 
of  the  two  ends  communicates  at  one  or  both  borders  of  the  cell 
substantiate  this  belief.  When  specimens  from  a  batch  of  eggs  are 
examined  from  day  to  day  a  change  takes  place  in  their  predominat- 
ing morphology,  as  the  cells  of  the  embtyo  tick  multiply.  The 
sharply  bipolar  forms  with  the  wide  intermediate  substance  gradu- 
ally become  fewer  and  fewer,  and  the  presence  of  some  which  have 
become  abnormally  large  and  swollen  indicates  that  many  are  in 
process  of  dissolution.  On  the  other  hand  the  number  of  smaller 
"diplococcoid"  forms  undergoes  an  increase,  and  many  of  them 
eventually  appear  in  groups  within  the  cytoplasm  of  the  embryonic 
cells  of  the  tick.  The  growth  of  the  latter,  however,  hardly  com- 
pensates for  the  disappearance  of  the  larger  bipolar  forms,  and  the 
impression  is  gained  that  the  total  number  of  organisms  has  under- 
gone a  decrease.  In  spite  of  this  change  the  virulence  of  the  eggs 
does  not  decrease.     In  one  batch  in  which  five  eggs  produced  infec- 


402  Contributions  to  Medical  Science 

tion  when  freshly  layed,  the  same  number  was  equally  virulent 
at  a  later  period,  when  the  total  number  of  organisms  had  appar- 
ently undergone  a  great  decrease  and  none  remained  but  the  smaller 
**diplococcoid"  bodies,  which  for  the  most  part  were  intracellular. 
This  series  of  observations,  which  has  been  repeated  many  times, 
leaves  the  impression  that  the  cells  with  the  large  amount  of  inter- 
polar  substance  are  involution  forms,  whereas  the  shorter  "diplo- 
coccoid"  bodies  and  the  solid  staining  cells  represent  the  more 
active  proliferating  organism. 

Examination  of  the  alimentary  sac,  salivary  glands,  and  oviduct 
disclosed  the  presence  of  large  numbers  of  similar  organisms,  but 
here  the  "proliferating"  forms  are  much  more  numerous  than  the 
so-called  involution  forms.  These  organs,  in  a  certain  sense, 
represent  free  surfaces  and  render  possible  the  removal  of  the 
products  of  bacterial  metabolism  and  a  renewal  of  food  substances 
for  the  micro-organisms.  The  eggs,  on  the  other  hand,  are  closed 
sacs,  covered  with  a  mucilaginous  substance,  which  renders  impos- 
sible the  separation  of  deleterious  and  inhibiting  material;  when 
the  bacterial  proHferation  has  reached  a  certain  degree,  further 
growth  may  also  be  limited  because  of  exhaustion  of  the  food  supply 
by  the  growing  embryo.  Thus  it  is  conceived  that  only  the  younger 
and  more  resistant  bacilli  survive,  and  appear  in  the  embryonic 
cells  and  later  in  various  tissues  of  the  active  tick. 

That  a  specific  agglutination  reaction  can  be  obtained  with  the 
organisms  as  they  exist  in  the  eggs  has  been  reported  previously." 
From  fifty  to  one  hundred  eggs,  depending  on  the  number  of 
organisms  they  contain,  are  crushed  in  about  1/20  c.c.  of  salt  solu- 
tion, and  the  suspension  shaken  until  the  distribution  of  the  bacilli 
is  uniform.  On  account  of  the  small  quantity  of  material  avail- 
able it  is  necessary  to  use  the  hanging-drop  method,  and  since  the 
emulsion  is  so  dense  in  refractile  elements  from  the  eggs,  determi- 
nation of  the  result  cannot  be  made  with  the  moist  preparation. 
After  two  hours'  residence  in  the  thermostat,  the  cover  glasses  are 
removed,  the  drops  permitted  to  dry,  after  which  they  are  fixed 
in  absolute  alcohol  and  stained  with  the  solution  of  Giemsa.  I 
may  quote  the  results  from  the  article  mentioned  above: 

"The  serum  of  the  normal  guinea-pig  either  causes  no  aggluti- 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    403 

nation  at  all,  or,  at  the  most,  produces  only  slight  agglutination 
in  proportions  of  i  to  i  and  i  to  20.  Dilutions  higher  than  this 
cause  no  agglutination.  In  testing  the  agglutinating  powers  of 
immune  serums,  three  animals  which  had  been  infected  from  dif- 
ferent sources,  and  had  recovered,  were  used.  One  (1751)  had  been 
infected  with  a  dermacentor  from  Idaho;  another  (1692)  with  a 
strain  handed  down  direct  from  guinea-pig  to  guinea-pig  for  nearly 
three  years  without  the  intervention  of  ticks,  the  original  infection 
having  been  obtained  from  the  blood  of  man;  the  third  (1757) 
with  a  strain  kept  in  the  same  way  since  last  spring.  Graded 
dilutions,  beginning  with  i  to  i  and  going  as  high  as  i  to  400,  were 
used  in  the  different  series,  with  the  striking  result  that  a  complete 
agglutinating  power  was  present  in  the  three  immune  serums  in 
dilutions  up  to  i  to  320.  It  was  somewhat  less  in  a  dilution  of 
I  to  400.  The  highest  dilution  which  will  cause  clumping  has  not 
been  ascertained. 

"No  fresh  immune  serum  from  man  is  at  hand,  but  tests  were 
made  with  three  specimens  which  are  about  five,  seven,  and  nine 
months  old,  respectively.  They  have  been  preserved  in  the  ice- 
chest  with  the  addition  of  0.3  per  cent  of  chloroform.  The 
peculiar  phenomenon  of  failure  to  agglutinate  in  concentrated 
solution  was  noted  with  all  three.*  With  the  oldest  serum  no  agglu- 
tination occurred  until  the  dilutions  of  i  to  320  and  i  to  400  were 
reached,  when  incomplete  clumping  was  produced.  With  the 
second  there  was  no  agglutination  in  the  dilution  of  i  to  i ,  distinct 
clumping  in  i  to  20,  i  to  40,  and  i  to  80,  with  little  or  none  in  higher 
dilutions.  In  the  serum  of  five  months'  standing  the  reaction  was 
absent  in  the  dilutions  of  i  to  i ,  i  to  20,  and  i  to  40,  positive  but 
not  complete  in  i  to  80,  i  to  160,  and  i  to  240,  with  very  little 
clumping  in  i  to  320,  and  i  to  400.  Normal  human  serum  caused 
clumping  in  a  dilution  of  i  to  i,  a  very  slight  amount  in  i  to  20, 
and  none  at  all  in  the  higher  dilutions." 

These  experiments  have  now  been  repeated  many  times  with 
invarying  results,  in  one  instance  distinct  agglutination  being 
obtained  with  the  immune  serum  in  a  dilution  of  i  in  1,000. 

The  result  of  the  agglutination  test,  then,  is  a  distinct  one. 

*The  "  agglutinoid  "  phenomenon. 


404  Contributions  to  Medical  Science 

The  blood  of  the  immune  animal  contains  agglutinins  in  high 
concentration  for  the  bacillus,  whereas  these  bodies  are  almost 
entirely  absent  from  the  serum  of  the  normal  animal.  Hence, 
in  the  light  of  experience  we  must  either  conclude  that  the  organism 
is  present  in  the  body  of  the  animal  during  his  attack,  or  suffer  our 
faith  in  the  value  of  the  agglutination  test  for  diagnostic  purposes 
to  be  shaken. 

Having  outlined  the  observations  which  favor  the  position  of 
the  bacillus  in  question,  I  may  refer  to  other  findings,  which 
apparently  throw  some  discredit  on  it. 

Scientific  proof,  among  other  things,  would  seem  to  demand 
that  the  bacillus  be  found  constantly  in  all  virulent  ticks  and 
eggs,  and  that  it  be  absent  constantly  from  all  non-virulent  material. 
In  the  early  stage  of  this  study  eggs  from  ticks  which  had  been 
definitely  proved  to  be  non-virulent  were  not  at  hand,  but  in  view 
of  the  presence  of  the  organism  in  the  internal  organs  of  the  viru- 
lent tick,  it  was  considered  that  the  tissues  of  non-virulent  ticks 
would  serve  as  satisfactory  controls  in  the  place  of  normal  eggs. 
It  so  happened  that  in  the  material  then  available  for  study  no 
bacilli  could  be  discovered  in  the  salivary  glands,  genital  organs, 
and  alimentary  sac  of  normal  ticks,  and  the  results  were  considered 
as  satisfactory  and  confirmatory. 

Later,  when  more  material  was  available,  and  particularly  when 
the  eggs  from  non-virulent  ticks  were  obtained,  it  was,  to  say 
the  least,  a  cause  for  astonishment,  that  bacilli  of  exactly  similar 
morphology  were  found,  often  in  great  abundance,  in  both  eggs  and 
tissues  which  experiments  showed  to  be  absolutely  without  viru- 
lence. Continued  observations,  indeed,  showed  that  an  organism 
of  this  character  is  present  with  almost  perfect  constancy  in  the 
eggs  and  tissues  of  Dermacentor  venustus  and  D.  modestus, 
regardless  of  their  infectivity.  In  only  occasional  individuals  has 
it  been  impossible  to  find  them. 

Not  only  do  they  resemble  exactly  the  bacilli  found  in  virulent 
eggs  and  tissues  in  their  morphology,  but  they  respond  to  the  specific 
agglutination  test  in  the  same  manner,  i.e.,  they  are  agglutinated 
by  immune  spotted  fever  serums  in  high  dilutions  and  not  by  normal 
serums,  except  in  dilution  of  i-io  or  1-20.      Therefore  on  the  basis 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    405 

of  morphology  and  the  agglutination  test  there  seems  to  be  no 
question  but  that  the  bacilli  found  in  avirulent  eggs  are  identical 
with  those  found  in  the  virulent.* 

The  situation,  therefore,  is  a  perplexing  one.  In  favor  of  the 
causative  relationship  of  the  bacillus  are  the  following  two  points: 
first,  the  presence  in  infected  blood  of  forms  which  correspond  in 
their  morphology  with  those  seen  in  the  eggs  and  tissues  of  the 
tick;  and,  second,  the  positive  agglutination  reaction  with  immune 
serums  in  contrast  with  a  negative  result  with  normal  serums. 
Against  it  is  the  fact  that  organisms  of  similar  morphology,  which 
respond  to  the  agglutination  reaction  in  the  same  manner,  are 
found  in  the  tissues  and  eggs  of  the  non-virulent  ticks. 

One  may  entertain  one  of  three  hypotheses  as  affording  an 
interpretation  of  the  results.  It  may,  in  the  first  place,  be  assumed 
that  the  organism  described  is  merely  a  constant  associate  of  the 
true  virus  in  the  infected  animal,  and,  consequently,  although  not 
having  a  necessary  relationship  to  the  disease,  it  may  cause  the 
formation  of  agglutinating  substances.  An  example  of  this  con- 
dition apparently  exists  in  hog  cholera,  in  which  the  true  virus  is 
very  minute  and  filterable,  whereas  the  hog  cholera  bacillus  is 
only  a  more  or  less  constant  associate  in  the  general  infection. 
Against  such  an  assumption  in  this  case,  however,  are  the  facts 
that  the  virus  of  spotted  fever  is  not  filterable,  and  in  the  perfectly 
fresh  eggs,  in  which  there  is  the  least  possible  morphological  con- 
fusion, the  bacillus  described  is  the  only  organism  which  can  be 
recognized.  Or,  second,  one  may  suppose  that  for  some  unknown 
reason  the  agglutination  reaction  is  not  specific  in  this  case,  and  that 
the  clumping  of  the  bacilli  by  the  immune  serum,  in  contrast  to 
the  negative  action  of  normal  serum,  is  brought  about  by  some 
pecuhar  and  unusual  conditions  in  the  experiments  as  they  were 
performed.  The  possibility,  however,  does  not  seem  sufl&ciently 
strong  to  shake  our  faith  in  the  time-honored  specificity  of  the  agglu- 
tination reaction.  Or,  finally,  it  may  be  assumed  that  the  bacillus 
in  question  is  the  true  cause  of  spotted  fever,  and  that  it  has  a 
tendency  to  undergo  a  loss  of  virulence  as  a  consequence  of  long 

*  Such  bacilli  could  not  be  found  in  the  eggs  and  tissues  of  specimens  of  Ornithodorus  meenini, 
Amblyoma  americanum,  and  an  eastern  Dermacentor  [variabilis  (?)]. 


4o6  Contributions  to  Medical  Science 

residence  in  the  tissues  of  the  tick,  thus  explaining  the  non-infectiv- 
ity  of  the  eggs  and  tissues  of  "normal"  ticks  which  are  rich  in 
similar  micro-organisms.  Such  an  assumption  carries  with  it  the 
idea  that  the  virus  of  spotted  fever  in  an  attenuated  form  has  an 
exceedingly  wide  distribution  in  the  ticks  of  the  Rocky  Mountain 
states,  and  that  it  assumes  virulence  only  under  certain  conditions, 
such  as  might  be  attained  by  sufficiently  frequent  passage  through 
some  suitable  animal.  If  a  virulent  tick  and  its  progeny  do  not, 
within  a  certain  period,  feed  on  a  susceptible  animal  and  convey 
spotted  fever  to  the  latter,  it  is  possible  that  the  strain  of  micro- 
organisms which  they  contain  becomes  hopelessly  avirulent  and 
incapable  of  producing  infection  in  any  animal  save  the  tick  itself. 
If,  on  the  other  hand,  they  do  convey  the  disease  to  susceptible 
animals  before  the  strain  has  lost  its  virulence,  the  organisms  may 
again  be  acquired  by  other  ticks,  and  the  period  of  pathogenicity 
thus  prolonged.  Indeed,  scarcely  any  fact  is  better  known  among 
bacteriologists  than  the  loss  of  virulence  which  a  micro-organism 
may  undergo  for  one  species  of  animal  as  a  consequence  of  repeated 
passage  (long  residence)  in  another  species,  and  the  frequency  with 
which  various  pathogenic  organisms  lose  virulence  for  a  host  in 
the  absence  of  occasional  passage  through  the  host  is  known  to 
all.  Further,  I  may  call  your  attention  to  evidence  already  cited 
that  strains  of  the  spotted  fever  virus  showing  differences  in  viru- 
lence actually  do  exist  in  nature,  as  illustrated  by  the  low  virulence 
of  the  Idaho  strains  for  both  man  and  the  guinea-pig  in  contrast 
with  the  high  virulence  of  the  virus  of  Montana.  Reasonably, 
therefore,  strains  of  still  less  or  no  virulence  for  these  animals 
may  exist  under  natural  conditions. 

As  a  working  basis,  then,  I  have  chosen  the  last  assumption 
as  being  the  most  reasonable  and  in  best  accord  with  the  known 
facts.  On  the  one  hand  it  is  hoped  that  the  bacillus  may  yet  be 
cultivated,  in  spite  of  many  failures  up  to  the  present  time,  after 
which  proof  of  its  position  may  be  more  easily  obtained.  At  the 
same  time  another  type  of  experiments  is  being  carried  on,  which, 
there  is  reason  to  believe,  will  be  equally  decisive  as  to  the  rela- 
tionship of  the  bacillus.  The  possibihty  of  vaccination  against 
spotted  fever  has  already  been  determined,  and  the  method  of 


Some  Aspects  of  Rocky  Mountain  Spotted  Fever    407 

sero-vaccination  has  been  described."  Successful  vaccination, 
not  hitherto  reported,  has  been  accompUshed  by  the  injection  of 
tissues  of  eggs  of  virulent  ticks  (rich  in  bacilli)  after  the  material 
has  been  sterilized  either  by  desiccation  or  by  chloroform.  If 
the  micro-organism  under  discussion  is  the  cause  of  spotted  fever, 
and  if  the  bacilli  found  in  a  virulent  material  are  merely  attenuated 
strains  of  the  virulent  organism,  equal  success  may  be  attained  in 
vaccination  with  the  latter.  Although  this  has,  indeed,  been 
accomplished  quite  recently,  it  seems  desirable  to  await  the  study 
of  a  more  abundant  material  before  submitting  further  conclusions. 

Prevention  and  serum  therapy. — ^To  go  into  detail  regarding 
methods  of  prophylaxis  and  control  of  spotted  fever,  which  have 
been  evolved  on  the  basis  of  the  results  cited,  would  be  to  trespass 
too  greatly  on  your  time.  An  account  of  the  procedures  recom- 
mended is  published  in  a  Report  to  the  State  Board  of  Health  of 
Montana,  previously  referred  to. 

Serum  therapy,  as  practiced  with  an  immune  serum  derived 
from  the  horse,  has  not  afforded  decisive  results,  and  perhaps  for 
several  theoretical  reasons.  In  the  first  place,  a  degree  of  immunity 
in  the  horse  greater  than  that  established  by  simple  recovery 
from  the  infection  cannot  be  obtained  until  the  organism  can  be 
cultivated  and  injected  in  excessive  quantities.  The  repeated 
injection  of  virulent  blood  into  an  immune  animal  is  of  little  or  no 
value  in  increasing  the  quantity  of  protective  antibodies  because 
of  the  comparatively  small  quantity  of  virus  which  such  blood 
contains.  The  immune  serum  is  of  such  strength  that  from 
0.3  to  0.5  c.c.  protects  against  i.o  c.c.  of  virus  (approximately 
1,000  pathogenic  doses).  Second,  experiments  with  animals  have 
shown  that  the  serum  cannot  be  expected  to  exert  a  marked  cura- 
tive effect  when  it  is  given  later  than  the  second  or  third  day  after 
inoculation,  and  even  ^in  this  case  such  an  amount  is  required 
as  to  reach  an  almost  prohibitive  quantity  when  translated  into 
terms  of  the  human  body  weight.  Further,  it  is  very  exceptional 
to  obtain  cases  of  spotted  fever  earlier  than  the  third  to  the  fifth 
day  of  sickness,  i.e.,  until  the  eruption  renders  the  diagnosis  reason- 
ably certain.  Third,  it  is  probable,  though  not  a  demonstrated 
fact,  that  the  serum  is  bactericidal  rather  than  antitoxic  in  its 


4o8  Contributions  to  Medical  Science 

nature.  If  this  proves  to  be  correct,  experience  with  other  similar 
serums  augurs  a  low  curative  value  regardless  of  the  concentra- 
tion of  germicidal  antibodies. 

No  unfavorable  effects,  so  far  as  can  be  determined,  have 
attended  its  use.  The  advisability  of  modifying  the  technic  of 
administration  so  that  large  quantities  are  given  intravenously 
early  in  the  course  of  infection  is  still  a  subject  for  consideration. 

REFERENCES. 

1.  Med.  Sent.,  1908,  16,  p.  704. 

2.  Ibid.,  1899,  7,  p.  433. 

3.  Jour.  Infect.  Dis.,  1904,  i,  p.  31. 

4.  Bull.  No.  14,  Hyg.  Lab.  U.S.  Pub.  Health  and  Mar.  Hosp.  Serv. 

5.  Bull.  No.  20,  ibid. 

6.  Fourth  Biennial  Report,  State  Board  of  Health  of  Montana,  1907-8,  p.  137. 

7.  Jour.  Infect.  Dis.,  1904,  i,  p.  31. 

8.  Jour.  Am.  Med.  Assn.,  1907,  49,  p.  84. 

9.  Fourth  Biennial  Report  of  State  Board  of  Health  of  Montana. 

10.  Jour.  Am.  Med.  Assn.,  1909,  52,  p.  379. 

11.  Jour.  Infect.  Dis.,  1908,  5,  p.  220. 


UNFINISHED    EXPERIMENTS    OF    DR.    HOWARD    T. 

RICKETTS    ON    ROCKY    MOUNTAIN 

SPOTTED    FEVER. 

Benjamin  Franklin  Davis. 

(From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

Following  the  death  of  Dr.  Ricketts  it  became  necessary  to 
discontinue  the  work  on  Rocky  Mountain  spotted  fever  pursued  in 
this  laboratory  under  his  direction.  A  number  of  problems  were 
being  studied  but  only  in  the  case  of  a  few  were  the  results  obtained 
such  that  conclusions  might  be  drawn  from  them.  It  is  the  pur- 
pose here  to  mention  the  various  paths  along  which  the  research 
was  progressing  and  to  indicate  the  conditions  obtaining  in  each 
when  the  work  was  stopped.  This  paper  does  not  include,  however, 
investigations  which  had  been  brought  to  a  reasonable  degree  of 
conclusion. 

I.     RESULT  OF  ATTEMPTS  TO  CULTIVATE  THE  VIRUS  OF  ROCKY 
MOUNTAIN   SPOTTED   FEVER. 

It  seems  quite  definitely  to  have  been  shown  that  the  virus  of 
Rocky  Mountain  spotted  fever  cannot  be  maintained  on  any  of 
the  ordinary  culture  media  at  room  or  incubator  temperature, 
aerobically  or  anaerobically  or  under  a  partial  oxygen  pressure 
such  as  may  be  obtained  by  the  method  used  by  Novak  in  his 
study  of  the  bacillus  of  Bang.''  Virus  added  to  uncoagulated 
egg-white  (hen's  egg)  or  to  yolk  of  egg,  or  to  a  mixture  of  white 
and  yolk,  and  left  at  room  temperature,  gradually  loses  its  infec- 
tivity  for  guinea-pigs  in  the  course  of  four  or  five  days.  Various 
serum  and  blood  media  have  failed  to  give  positive  results  as  have 
also  media  containing  a  filtrate  of  various  bacterial  cultures.  It 
was  thought  that  possibly  the  virus  might  be  destroyed  in  vitro 
by  the  action  of  immune  bodies  contained  in  the  serum  and  the 
attempt  was  made  to  reduce  this  action  to  a  minimum  by  dilu- 
tion of  the  serum  with  salt  solution,  with  broth,  and  by  the  addition 

'  Ann.  de  I'Inst.  Pasteur,  1908,  22,  p.  541- 

409 


4IO  Contributions  to  Medical  Science 

of  serum  in  which  an  anticomplement  specific  for  guinea-pig 
complement  had  been  developed,  but  apparently  without  success. 
The  results  of  preliminary  experiments  with  the  intraperitoneal 
collodion  sac  method  of  cultivation  seem  to  indicate  that  the 
virus  whether  undiluted,  whether  suspended  in  salt  solution 
(0.85  per  cent),  in  ordinary  nutrient  broth  or  in  anti-complement 
serum,  loses  its  infectivity  very  rapidly.  That  the  virus  does 
not  pass  through  the  collodion  membrane  seems  certain  because 
(i)  guinea-pigs  in  which  sacs  containing  virus  have  been  placed 
do  not  contract  spotted  fever,  and  (2)  they  develop  the  disease  at 
subsequent  immunity  tests.  Clegg's^  reported  success  in  cultivat- 
ing B.  leprae  in  symbiosis  with  amebas  suggested  a  similar  experi- 
ment with  the  spotted  fever  virus.  Up  to  this  time,  however, 
no  substantial  progress  had  been  made. 

II.     DISAPPEARANCE  OF  ORGANISMS  FROM  THE  EGGS  AND  TISSUES 
OF   TICKS   MATURED   ON   IMMUNE   GUINEA-PIGS. 

Dr.  Ricketts  has  described^  (p.  36)  a  minute,  bipolar  body  which  is 
found  almost  constantly  in  the  eggs  and  tissues  of  normal  and 
infective  Idaho  and  Montana  ticks  (D.  modes tus  and  D.  venustus, 
respectively),  and  which  he  was  inclined  to  believe  bears  a  causal 
relationship  to  Rocky  Mountain  spotted  fever.  Granting  that  this 
bipolar  body  is  the  cause  of  the  disease,  the  assumption  was  made 
that  by  feeding  ticks  in  whose  tissue  such  bodies  were  present  on 
immune  guinea-pigs,  antibodies  would  be  absorbed  by  the  ticks 
from  the  pigs  and  would  destroy  the  supposed  bacteria,  with  the 
result  that  the  latter  would  disappear  from  the  tissues  of  the  ticks 
and  that  the  eggs  of  such  ticks  would  be  free  from  bacilli.  In 
accordance  with  this  idea  two  batches  of  bacilli-rich  larvae  were 
selected.  One  batch  was  matured  on  immune  guinea-pigs;  the 
other  batch  was  matured  on  normal  guinea-pigs  as  a  control 
experiment.  Although  this  experiment  is  unfinished,  the  indica- 
tions seem  to  be  that  there  is  a  progressive  diminution  in  the  num- 
ber of  organisms  in  the  tissue  and  eggs  of  the  ticks  of  both  batches. 
Actual  counts  have  not  been  made;  the  above  is  simply  the  opinion, 

'  Phil.  Jour.  Sc,  B.  Med.,  igog,  4,  p.  77. 
'Jour.  Am.  Med.  Assn.,  1909,  32,  p.  379- 


Unfinished  Experiments  of  Dr.  Howard  T.  Ricketts    411 

based  on  examination  of  the  material,  of  the  one  (Dr.  Maria  B. 
Maver)  who  was  carrying  out  the  experiment.  An  interpretation 
of  such  a  result  is  hardly  possible  at  this  time.  If  such  a  diminu- 
tion does  occur,  however,  several  explanations  suggest  themselves, 
viz.:  (i)  the  so-called  bacillus  may  bear  no  relation  to  spotted 
fever  but  may  be  some  form  which  is  picked  up  by  the  ticks  in 
nature  and  which  gradually  disappears  from  the  ticks  when  the 
latter  are  removed  from  their  natural  surroundings  and  hence 
from  the  source  of  supply;  (2)  the  bacillus  may  be  the  cause  of 
spotted  fever;  it  disappears  from  the  ticks  matured  on  immune 
pigs  in  accordance  with  the  considerations  outlined  above.  To 
explain  its  disappearance  from  ticks  matured  on  normal  pigs  we 
may  reason  as  follows:  The  ticks  used  were  non-infective,  which 
means  presumably  that  the  bacilli  which  they  contained  were 
avirulent,  that  is,  were  not  capable  of  proliferation  in  the  body 
of  a  guinea-pig.  If  this  were  the  case,  then  the  blood  of  a  normal 
guinea-pig  might  be  supposed  to  exert  an  influence  on  the  organ- 
isms similar  to  that  of  the  blood  of  an  immune  pig  with  the  result 
that  the  bacilli  would  tend  to  disappear  from  the  body  of  the  tick. 
This  explanation  seems  to  gain  some  support  from  the  results 
of  vaccination  experiments,  to  be  described  later,  in  which  suc- 
cessful vaccination  against  the  active  virus  was  obtained  by  the 
use  of  eggs  and  tissues  from  non-infective  but  bacilli-rich  ticks. 

III.     THE    PRESENCE   OF   INFECTED   OR  IMMUNE  ANIMALS  IN  NATURE. 

This  problem  was  assigned  to  Mr.  J.  J.  Moore  for  the  spring 
and  summer  of  19 10  in  the  hope  that  the  results  would  test  the 
hypothesis  of  Dr.  Ricketts  that  Rocky  Mountain  spotted  fever 
is  maintained  in  nature  by  a  continuous  cycle  of  passage  from 
infected  ticks  to  susceptible  animals  and  from  animals  thus  infected 
back  to  tick  larvae,  nymphs,  and  adults.  Infective  ticks  have 
been  found  in  nature;  it  has  been  shown  that  certain  of  the  smaller 
wild  animals  are  susceptible  to  the  disease  and  that  ticks  may  not 
only  acquire  the  virus  from  infected  wild  animals,  but  may  in  turn 
transmit  the  virus  to  other  animals.  The  finding  of  infected  or 
immune  animals  in  nature  would,  therefore,  complete  the  evidence. 
The  method  to  be  followed  was,  briefly,  to  capture  wild  animals, 


412  Contributions  to  Medical  Science 

to  inject  some  of  their  blood  into  normal  guinea-pigs  and  to  watch 
the  pigs  for  symptoms  of  spotted  fever.  In  case  spotted  fever 
failed  to  develop,  each  wild  animal  was  to  be  given  an  injection  of 
virulent  guinea-pig  blood  and,  after  a  suitable  interval,  again  bled 
for  injection  into  a  normal  guinea-pig.  The  production  of  spotted 
fever  in  the  first  guinea-pig  would  prove  that  the  wild  animal 
under  observation  was  suffering  with  the  disease.  Failure  to 
produce  spotted  fever  in  the  second  guinea-pig  would  prove  that 
the  wild  animal  was  immune  to  the  disease  and  had  therefore 
probably  suffered  from  it  at  some  previous  time.  It  would  be 
necessary  to  make  the  passage  into  the  guinea-pig  because  in  none 
of  the  susceptible  wild  animals  thus  far  tested  are  the  symptoms 
produced  by  the  spotted  fever  infection  sufl&cient  for  diagnosis. 
Unfortunately  the  experiments  outlined  could  not  be  carried  out. 

IV.      INFECTIVITY  OF  THE  EGGS  OF  INFECTIVE  AND  NON- 
INFECTIVE   TICKS 

The  technic  of  these  experiments  was  as  follows:  normal 
guinea-pigs  were  given  intraperitoneal  injections  of  an  emulsion 
in  salt  solution  (0.85  per  cent)  of  the  eggs  to  be  tested  and  their 
temperatures  taken  for  the  subsequent  ten  days.  A  week  or  two 
later,  an  immunity  test,  consisting  of  the  injection  of  i  c.c.  of 
fresh  virus,  i.e.,  blood  from  a  sick  guinea-pig,  was  given;  if  the 
animal  failed  to  develop  a  fever  after  the  egg  injection  and  became 
infected  in  his  immunity  test  or  if  he  did  develop  a  fever  follow- 
ing the  injection  of  the  eggs  and  still  was  not  immune  to  the 
active  virus,  the  result  of  the  experiment  was  declared  to  mean 
that  the  eggs  were  non-infective.  The  outcome  of  the  immunity 
test  then  furnished  the  basis  for  the  interpretation  of  results. 
At  first  a  series  of  guinea-pigs  was  used  in  each  experiment, 
dilutions  of  egg  emulsion  equivalent  to  5,  10,  20,  40,  60,  and  80 
eggs  being  injected  into  susceptible  animals.  Later,  and  this 
applies  to  the  majority  of  the  experiments,  a  uniform  dose, 
equivalent  to  50  eggs,  was  employed.  The  eggs  were,  on  the 
average,  less  than  one  week  old  when  the  experiments  were 
performed  and  had  been  left  in  the  dark,  at  the  temperature  of 
the  ice-chest,  since  they  were  laid. 


Unfinished  Experiments  of  Dr.  Howard  T.  Ricketts    413 

The  results  are:  Of  12  infective  Montana  females  (D. 
venustus)  the  eggs  of  3  produced  infection — 25  per  cent;  of  16 
non-infective  Montana  females  none  of  the  eggs  were  virulent; 
of  24  infective  Idaho  females  (D.  modes tus)  the  eggs  of  but 
3  were  virulent — 12|  per  cent;  none  of  the  eggs  of  the  23  non- 
infective  Idaho  ticks  which  were  tested  produced  the  disease. 
Eggs  from  63  of  the  batches  used  were  examined  microscopically 
for  bacilli  and  in  all  cases  typical  bipolar  organisms  were  found; 
12  batches  were  not  examined.  In  five  of  the  six  experiments 
which  resulted  positively  the  eggs  were  injected  in  quantities  vary- 
ing from  5  to  80  according  to  the  method  mentioned  above.  In 
all  such  cases  5  eggs  proved  sufficient  to  produce  infection. 

Work  on  the  infectivity  of  emulsions  in  salt  solution  of  larvae, 
nymphs,  and  the  tissues  (5)  of  infected  adult  ticks  was  begun  and 
some  positive  results  were  obtained  with  all  the  materials  which 
had  been  prepared.  The  experiments  are  too  few  in  number  to 
justify  more  extensive  recapitulation. 

The  results  of  these  experiments  seem  to  indicate  that  but  a 
small  proportion  of  infective  ticks  transmit  the  virus  through  the 
egg,  thus  supporting  the  contention  that  the  virus  is  kept  alive 
in  nature  by  passage  from  tick  to  wild  animal  and  from  wild 
animal  to  tick.  It  may  be  that  in  the  egg  the  virus  passes  through 
a  developmental  cycle  of  some  kind  in  certain  phases  of  which  it  is 
non-virulent,  or  possibly  the  technic  was  not  suitable  to  the  object 
of  the  experiment,  all  of  which  may  be  decided  only  by  further 
investigation.  In  a  measure,  the  results  seem  to  support  the  idea 
that  the  Idaho  virus  is  less  virulent  than  the  Montana  virus,  in 
that  there  were  but  half  as  many  positive  results  using  the  eggs  of 
the  Idaho  ticks  as  there  were  when  the  eggs  of  Montana  ticks  were 
injected.  Such  a  conclusion  on  the  evidence  at  hand,  however, 
must  be  regarded  as  tentative  only. 

V.      VACCINATION  WITH  TICK  EGGS  AND   TISSUES. 

In  one  of  his  last  communications'  Dr.  Ricketts  said:  "Suc- 
cessful vaccination,  not  hitherto  reported,  has  been  accomplished 
by  the  injection  of  the  tissues  and  eggs  of  virulent  ticks  (rich  in 

'  Med.  Rec.,  igog,  76,  p.  i. 


Date 

6/30 

7/1 

7/2 

Temperature 

103 

103.2 

Date 

7/8 

7/9 

7/10 

Temperature 

102 

101.6 

414  Contributions  to  Medical  Science 

bacilli)  after  the  material  has  been  sterilized  either  by  desiccation 
or  by  chloroform."  It  seems  desirable  to  supplement  this  state- 
ment by  the  publication  of  tj^^ical  experiments  and  the  tabulation 
of  the  results  obtained  up  to  the  time  when  the  work  was  discon- 
tinued. This  work  was  begun  by  Dr.  Ricketts  and  subsequently 
carried  on  at  various  times  by  Mr.  R.  M.  Wilder,  Dr.  Maria  B. 
Maver,  and  Mr.  W.  F.  Petersen. 

Experiment  i. — June  29,  1909.  Dried  eggs  of  Montana  female  No.  49.  They 
were  virulent  when  fresh  and  contained  bacilli.  They  had  been  dried  for  about  4 
months.  None  of  the  eggs  hatched,  although  the  embrj'os  had  begun  to  form.  354 
eggs  were  injected  intraperitoneally  into  guinea-pig  2244  after  they  had  been  ground 
up  in  salt  solution  in  an  agate  mortar. 

7/3  7/4  7/5  7/6  7/7 

103  102.4         101.6         102.6         102.2 

7/11  7/12  7/13  7/14  7/1S 

102.2         101.8         102.7         103.7         102 

First  immunity  test,  j-ij-'og — i  c.c.  virus  intraperitoneally. 

Date  7/18        7/19        7/20        7/21         7/22         7/23         7/24        7/25         7/26        7/27 

Temperature        loi         102.4      103.2         102         102.2       102.6       102.4       102.2       102.8       102.6 

Date  7/28         7/29         7/30         7/31  8/1  8/2  8/3  8/4  8/s 

Temperature      102.4      103.8      102.6        102        101.4       102.4        103         102.8       101.8 

Second  immunity  test,  ii-6-'o9 — i  c.c.  of  virus  was  injected  intraperitoneally. 
The  result  paralleled  that  of  the  first  immunity  test.  Therefore  vaccination  was 
successful,  the  virus  used  for  the  tests  being  potent  as  shown  by  the  production  of 
spotted  fever  in  the  control  animal. 

Experiment  2. — September  26,  1909.  D.  venustus,  female  190.  Infected  with 
"natural"  tick  strain  on  about  6-29-'o9;  tested  on  guinea-pig  7-26-'o9  and  found 
infective;  killed  and  dissected  9-20- '09:  Many  bacilli  found  in  ovary,  gut,  and  saUvary 
glands.  Nearly  all  of  the  salivary  gland  removed  for  staining.  Fair  portion  of 
ovary  removed,  and  a  small  part  of  gut.  Organs  triturated  in  salt  solution,  and  a 
drop  of  chloroform  added  to  sterilize.  After  two  or  three  daj's  placed  in  desiccator 
and  allowed  to  remain  there  in  a  partial  vacuum  for  three  days.  On  9-26  the  material 
was  re-emulsified  in  salt  solution  and  injected  intraperitoneally  into  guinea-pig. 

Date  9/27  9/28  9/29  9/30  lo/i  10/2  10/3  10/4  lo/s 

Teinp)erature     103.4        102.6        102.8  102  102.2        103.2        102.6        102.8  103 

Immunity  test — i  c.c.  virus  intraperitoneally  io-io-'o9. 

Date  lo/ii  10/12  10/13  10/14  10/15  10/16  10/17  10/18  10/19      10/20 

Temperature  102.  i  102.9  102.3  103.2  103.2  103.2  102.4  102.9       102.8 

Date  10/21  10/22  10/23  10/24  10/25  10/26  10/27  10/28  10/29 

Temperature  103.4  103          103  102.4  102.4        103  102.8  102.6  102.8 

A  second  immunity  test,  given  40  days  later  in  order  to  rule  out  a  possible 
passive  immunization  derived  from  the  injected  tick  tissues,  resulted  similarly  to  the 
first;  therefore  the  injection  of  sterilized  tissues  from  infected  ticks  appears  to  have 
induced  a  high  degree  of  immunity  in  guinea-pigs.  The  duration  of  this  immunity 
was  not  tested. 


Unfinished  Experiments  of  Dr.  Howard  T.  Ricketts    415 

The  serum  of  this  animal  appeared  to  possess  but  little,  if  any,  power  of  protecting 
other  animals  when  injected  simultaneously  with  large  doses  of  virus.  An  accurate 
immunological  analysis  was  not  attempted. 

In  the  same  communication  Dr.  Ricketts  said  further:  "If 
the  micro-organism  under  discussion  is  the  cause  of  spotted  fever, 
and  if  the  bacilH  found  in  avirulent  material  are  merely  attenuated 
strains  of  the  virulent  organism,  equal  success  may  be  attained 
in  a  vaccination  with  the  latter.  Although  this  has,  indeed,  been 
accomplished  quite  recently,  it  seems  desirable  to  await  the  study 
of  a  more  abundant  material  before  submitting  conclusions." 
The  technic  of  these  experiments  was  the  same  as  that  used  in 
the  work  with  the  material  from  ticks  whose  infectivity  had  been 
proven.  The  results  are  summed  up  in  the  accompanying  tables. 
These  tables  show:  (i)  that  practically  all  the  tick  material  used 
contained  bacilli;  (2)  of  the  experiments  with  the  eggs  of  Montana 
ticks  (D.  venustus)  33  per  cent  yielded  positive  results,  while  with 
the  tissues,  50  per  cent  were  positive;  of  the  experiments  with  the 
Idaho  ticks  (D.  modestus),  11  per  cent  were  positive,  while  with 
the  tissues  in  66  per  cent  vaccination  was  obtained ;  of  the  positive 
results  with  the  eggs  of  the  D.  venustus  33  per  cent  were 
obtained  with  the  eggs  of  non-infective  ticks  (avirulent  material) 
while  in  the  case  of  the  tissues  50  per  cent  of  the  positive  results 
were  obtained  with  virulent  material  and  50  per  cent  with  non- 
virulent  material;  of  the  positive  results  with  the  eggs  of  the  D. 
modestus,  all  (100  per  cent)  were  obtained  with  the  eggs  of  infect- 
ive ticks,  as  were  also  the  positive  results  obtained  through  the 
injection  of  tissues.  Although  these  experiments  must  be  regarded 
as  being  purely  of  a  preliminary  nature,  they  seem  to  indicate 
quite  strongly  that  vaccination  with  tick  eggs  and  tissues  is  pos- 
sible and  that  this  may  be  accomplished  by  the  use  of  material 
from  non-infective  ticks  (of  the  Montana  strain  at  least)  about  as 
readily  as  with  material  from  ticks  whose  infectivity  has  been  proven. 
This  would  seem  to  bear  out  the  suggestion  quoted  above  as  to 
the  possible  relationship  of  the  bacillary  forms  found  in  certain 
normal  and  in  apparently  all  infective  tick  eggs  and  tissues  to  spot- 
ted fever  and  to  each  other.     The  ideal  control  for  such  experiments 


4i6 


Contributions  to  Medical  Science 


— that  is,  the  use  of  eggs  and  tissues  which  are  free  from  bacilli — 
has  not  been  attempted. 

TABLE  I. 

Showing  the  Result  of  Experiments  in  Vaccinating  Guinea-Pigs  against  Spotted  Fever  by  the 

Injection  of  Tick  Eggs  and  Tissues. 


Ticks 

No.  Tests 
of  Eggs 
(Av.  No. 
Eggs  per 

Test  =400) 

No. 
Infec- 
tions 
from 
Eggs 

No.  Suc- 
cessful 
Vaccina- 
tions 

No.  Unsuc- 
cessful 
Vaccina- 
tions 

.0£ 
0  ">  S 

No.  Suc- 
cessful 
Vaccina- 
tions 

No.  Unsuc- 
cessful 
Vaccina- 
tions 

D.  venustus 

D.  modestus 

9 
19 

0 

I 

3  (33%) 
2  (11%) 

6 
16 

4 
3 

0 
0 

2  (so%) 
2  (66%) 

2 
I 

Total 

28 

I 

S  (i8.S%) 

22 

7 

4  (S7%) 

TABLE  2. 
Showtng  the  Relation  of  the  Bacill,ary  Content  of  Eggs  and  Tissues  to  the  Infectivity  of 
THE  Corresponding  Ticks — the  Ticks  Being  Tested  on   Normal  Guinea-PigS"  before  the 
Eggs  Were  Laid  and  before  Being  Dissected  for  the  Purpose  of  Obtaining  the  Tissues. 
The  Tissues  Used  Consisted  Usually  of  Ovary,  Salivary  Gland,  and  Gut. 


Ticks 

No.  Sets 
of  Eggs 

Which 
Contain 

Bacilli 

No.  Sets 
of  Eggs 
not  Ex- 
amined 

No.  Sets 
of  Eggs 

from 
Infective 

Ticks 

No.  Sets 
of  Eggs 
from  Non- 
infective 
Ticks 

No.  Sets 
of  Tissues 
Which 
Contain 
Bacilli 

No.  Sets 
of  Tissues 
not  Ex- 
amined 

No.  Sets 
of  Tissues 

from 
Infective 

Ticks 

No.  SeU 
of  Tissues 
from  Non- 
infective 
Ticks 

D.  venustus 

D.  modestus.  . . . 

8 
i8 

I 
I 

5                  4 
7                12 

4 
3 

0 
0 

2 
3 

2 
0 

ToUl 

26 

2 

12                16 

7 

0 

S 

2 

TABLE  3- 
Showing  the  Relation  of  the  Vaccinating  Power  of  Eggs  and  Tissues  to  the  iNFEcnviTY  op 

THE  Corresponding  Ticks. 


Ticks 

No.  Positive 
Results 
FoUowing 
Injection  of 
Eggs  of  In- 
fective 
Ticks 

No.  Positive 

F^rng        Tot^^No. 
Injection  of         ^°^^^J^ 
Eggs  of  Non-!    JthEek 

infective         ^^^°  ^^gs 
Ticks 

Total  No. 

Positive 

Results 

with  Tissues 

No.  Positive 

Results 

Following 

Injection  of 

Tissues  of 

Infective 

Ticks 

No.  Positive 

Results 

Following 

Injection  of 
Tissues  of 

Non-infect- 
ive Ticks 

D.  venustus 

D.  modestus 

1  (33i%) 

2  (100%) 

2  (66?%) 
0 

3 
2 

2 
2 

1  (so%) 

2  (100%) 

I  (So%) 
0 

Total 

3 

2 

5 

4 

3 

VI.     ALTERATIONS  IN  THE  VIRULENCE  OF  SPOTTED  FEVER  VIRUS  IN 
THE   BODIES   OF   TICKS. 

From  the  much  lower  death  rate  among  the  patients  infected 
with  the  spotted  fever  of  Idaho,  as  compared  with  that  of  those 
suffering  from  the  Montana  disease;  from  the  much  milder  clini- 
cal course  of  the  former;    and  from  the  fact  that  in  1908  it  had 


Unfinished  Experiments  of  Dr.  Howard  T.  Ricketts    417 

been  found  impossible  to  keep  the  Idaho  disease  going  in  the 
guinea-pig  by  successive  passages,  the  conclusion  seemed  to  be 
warranted  that  the  Idaho  virus  was  less  virulent  for  man  and  for 
the  guinea-pig  than  the  Montana  virus — the  probable  identity  of 
the  two  having  been  established  by  the  similarity  of  clinical  mani- 
festations and  by  the  results  of  immunity  tests.  The  question 
now  arose,  assuming  that  the  viruses  are  identical,  why  this  dif- 
ference in  virulence  ? 

It  had  been  shown  that  the  Idaho  tick  could  acquire  and  trans- 
mit the  spotted  fever  of  Montana  and  that  the  Montana  tick 
could  acquire  and  transmit  the  spotted  fever  of  Idaho.  As  these 
ticks  differ  slightly  from  each  other  morphologically  it  was  assumed 
that  other  differences  might  be  present  of  such  a  nature  as  to 
alter  the  spotted  fever  virus  which  happened  to  become  resident 
in  the  tissues,  thus  accounting  for  the  difference  in  the  virulence 
of  the  two  strains.  Experimental  demonstration  was  attempted 
as  follows: 

Alteration  of  Virulence  of  Spotted  Fever  Virus  in  the  Idaho  Tick. 

The  plan  was  to  infect  Idaho  ticks  (D.  modes tus)  by  allowing 
them  to  feed  on  guinea-pigs  which  were  infected  with  the  Montana 
spotted  fever,  and  to  keep  the  disease  going  in  them  through 
inheritance  for  as  long  a  period  as  need  be. 

As  normal  guinea-pigs  were  infected  by  these  ticks,  consecutive 
passage  was  to  be  made  from  such  guinea-pigs  through  a  series  of 
7  to  10  generations  in  order  to  determine  whether  or  not  the  disease 
would  "run  out"  in  the  guinea-pig,  this  being  taken  as  a  test  of 
virulence,  based  on  the  experience  of  1908. 

Idaho  Disease  in  Montana  Ticks  (D.  venustus). 

The  primary  object  of  this  experiment  was  to  determine  whether 
residence  of  the  Idaho  spotted  fever  in  the  Montana  ticks  would 
result  in  an  increase  of  the  virulence  of  the  Idaho  disease.  The 
test  of  increase  of  virulence  would  be  the  possibility  of  keeping 
the  disease  alive  by  continuous  passage  in  the  guinea-pig  after  it 
had  been  in  the  Montana  ticks. 

Regarding  the  first  of  these,  the  Montana  fever  in  the  Idaho 


41 8  Contributions  to  Medical  Science 

tick,   but   three  experiments  were   carried  through,   of  which  a 
brief  statement  follows: 

1.  Duration  of  residence  of  virus  in  the  tick,  approximately  19 
days;  infected  guinea-pig  on  September  12,  1909.  Successive 
passages  were  made  into  guinea-pigs  in  each  case  on  the  third  day 
of  fever;  all,  excepting  one,  died.  There  was  no  decrease  in  viru- 
lence manifest. 

2.  Duration  of  residence  of  virus  in  tissues  of  the  tick  was 
much  shorter,  since  the  transfer  from  infecting  pig  to  test  pig  was 
immediate.  The  second  pig  ran  a  typical  course  of  spotted  fever 
and  successive  passages  were  made  from  him  into  the  seventh 
generation  of  pigs.  All  of  these  pigs  died  except  the  last  which 
was  killed  for  other  purposes.  The  infection  appeared  to  be  as 
virulent  as  the  Montana  disease  ever  had  been. 

3.  The  eggs  of  this  tick  were  injected  into  guinea-pig  on  March 
3,  1909.  Residence  of  virus  in  tissues  of  the  tick  about  two  months. 
The  guinea-pig  had  a  typical  attack  of  spotted  fever;  consecutive 
passages  were  made  into  the  fifth  generation  of  guinea-pigs  with 
no  sign  of  a  decrease  in  virulence. 

In  so  far  as  the  experiments  were  carried,  then,  no  alteration 
in  the  virulence  of  the  Montana  virus  in  the  Idaho  tick  was  demon- 
strated. 

Concerning  the  second  line  of  experiments — the  Idaho  disease 
in  the  Montana  ticks — no  work  has  been  done,  because  the  Idaho 
strain  obtained  from  the  patient  "Spaniard,"  which  was  the  only 
one  available  at  the  time,  proved  to  be  of  such  virulence,  con- 
trary to  previous  experience  with  the  Idaho  strains,  that  it  could 
in  the  first  instance  be  kept  alive  by  passage,  thus  destroying  the 
criterion  for  determining  the  increase  of  virulence. 


COMPLEMENT  DEVIATION  IN  ROCKY  MOUNTAIN 
SPOTTED   FEVER.^ 

Benjamin  F.   Davis  and  William  F.   Petersen. 

{From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

The  experiments  here  described  have  been  made  at  various 
times  in  this  laboratory,  the  original  object  being  to  provide  a  basis 
for  an  early  diagnostic  test  for  Rocky  Mountain  spotted  fever. 

The  first  experiments  were  carried  out  by  Liborio  Gomez  at  the 
suggestion  of  Dr.  Ricketts  and  we  shall  give  a  brief  outline  of  his 
unpublished  results.  Gomez  used  an  ox-rabbit  system,  using  the 
serum  from  infected  guinea-pigs  as  antigen  (drawn  during  second 
to  fourth  day  of  fever)  and  immune  serum  from  guinea-pigs  and 
rabbits  as  antibody.  He  first  determined  that  the  serum  contain- 
ing the  antigen  and  the  serum  containing  the  antibody  possessed 
no  hemolytic  amboceptors  for  ox  corpuscles.  His  experiments 
then  followed  the  method  of  Bordet-Gengou.  A  positive  devia- 
tion was  obtained  in  the  case  of  one  guinea-pig  which  had  recovered 
from  the  disease  and  which  had  received  an  immunity  test  some 
ten  weeks  before  being  killed.  In  a  recovered  rabbit  he  also  obtained 
a  positive  though  weak  fixation  of  the  complement.  In  two  guinea- 
pigs  killed  four  or  five  months  after  recovery  from  a  single  attack 
of  spotted  fever  Gomez  found  a  retardation  of  hemolysis  of  from 
two  to  five  hours,  as  compared  with  controls  of  immune  serum  and 
virus  alone. 

In  his  experiments  with  the  immune  guinea-pig  serum  Gomez 
left  the  tubes  containing  the  complement-antibody-antigen  mixture 
in  the  incubator  for  over  14  hours  before  adding  the  hemolytic 
system.  This  period  of  time  is  long  and  leaves  some  doubt  as  to 
the  value  of  the  result  obtained.  Gomez  notes  that  the  smaller 
doses  of  antigen-antibody  recommended  by  Bordet  and  Gengou 
failed  to  give  results.  In  his  experiments  with  the  blood  of  a  re- 
covered rabbit  he  established  a  binding  with  0.6  c.c.  of  the  serum. 
Gomez  notes,  however,  that  large  doses  of  antibody  (1-2  c.c.)  will, 

•  From  Jour.  Infect.  Dis.,  igix,  8,  p.  330. 

419 


0 


420  Contributions  to  Medical  Science 

when  mixed  with  complement,  bind  or  destroy  its  action  without 
the  presence  of  antigen. 

With  the  idea  of  further  testing  this  reaction,  Dr.  Ricketts  and 
Mr.  R.  M.  Wilder  made  several  experiments,  using  the  eggs  of 
infected  ticks  as  antigen.     It  has  already  been  shown  that  the  eggs  i 

of  infected  ticks  are  rich  in  an  organism  which  is  supposedly  the 
cause  of  Rocky  Mountain  spotted  fever  and  it  was  supposed  that 
these  eggs  would  therefore  afford  a  suitable  source  of  antigen.  In 
these  experiments  a  goat-rabbit  hemolytic  serum  was  used  and  the 
exact  dosage  of  fresh,  normal  guinea-pig  complement  was  fixed  for 
each  experiment. 

In  the  first  three  experiments  of  the  series  eggs  were  used  in 
quantities  varying  from  i  to  loo,  the  antibody  being  varied  from 
0.05  to  0.1  c.c.  As  an  example  of  these  experiments  we  insert 
Table  i. 

From  these  experiments  it  will  be  noted  in  Group  A  (Table  i) 
that  there  is  a  shght  decrease  in  hemolysis  as  the  number  of  eggs  is 
increased.  In  Group  B  with  a  larger  amount  of  antibody  the  same 
result  is  noted,  although  the  total  amount  of  hemolysis  is  greater 
in  each  corresponding  tube  than  in  Group  A.  The  degree  of  hemol- 
ysis is  not  dependent  on  the  number  of  eggs,  since  Group  C  shows 
a  constant  amount  of  hemolysis  in  each  tube.  Evidently  there  was 
some  reactivation^  of  the  heated  serum  by  the  unheated,  and  this 
reactivation  was  greater  when  the  amount  of  heated  complement 
in  the  antibody  (guinea-pig  serum)  was  greater.  This  was  borne 
out  by  the  results  in  tubes  17  and  18  in  which  a  large  amount  of 
antibody  increased  the  hemolytic  action  of  o.oi  c.c.  of  complement. 
In  this  experiment  the  total  amount  of  complement  used  was  not 
quite  sufiicient  to  cause  complete  lysis,  as  shown  in  tube  13. 

In  the  second  experiment  of  Ricketts'  series  larger  numbers  of 
eggs  were  used  (75-100);  the  results  were  again  inconclusive.  It 
was  found  that  the  eggs  alone,  without  the  addition  of  antibody, 
were  capable  of  preventing  lysis.  It  was  again  noted  that  antibody 
and  complement,  when  together,  were  more  hemolytic  than  com- 

■  That  such  an  activation  takes  place  was  shown  in  an  experiment  in  which  we  first  established  the 
minimum  dosage  of  normal  complement  and  then  added  varying  amounts  of  antibody  to  a  subminimal 
dose  of  complement.  Doses  of  inactivated  antibody  from  o.oi  c.c.  up  caused  complete  laking  in  the 
tubes,  although  alone  the  antibody  had  absolutely  no  effect. 


Complement  Deviation  in  Spotted  Fever 


421 


plement  alone,  and  it  was  assumed  that  the  antibody  contained 
hemolytic  amboceptors  which  were  activated  by  the  complement. 
An  effort  was  made  in  the  third  experiment  to  absorb  the  hemolytic 
amboceptor  supposed  to  be  present  in  the  antibody,  but  the  results 


TABLE  I. 
CoMPi£MENT  Deviation  in  Spotted  Fever. 


Groups 

Group  A 

I 

2 

3 

4 

Group  B 

S 

6 

7 

8 

Group  C 

9 

10 

II 

12 

Group  D 

13 

14 

Group  E 

IS 

16 

Group  F 

17 

18 

Group  G 
19 


Sensitized 
Corpuscles 


Antibody 

Antigen 
(Eggs) 

0.0s 

I 

0.0s 

10 

0.0s 

20 

0.05 

5° 

0.1 

1 

0.1 

10 

0.1 

20 

0.1 

SO 

0 

I 

0 

10 

0 

20 

0 

50 

0 

0 

0 

0 

0.0s 

0 

0.1 

0 

0.0s 

0 

0.1 

0 

0 

0 

0 

0 

Complement 


Results 


o-S 
o.s 
o-S 
o.s 

o.s 
0.5 
o.s 
o.s 

o.s 
o.s 
o.s 
o.s 

o.s 
o.s 

o.s 
o.s 

o.s 
o.s 


Unsensitized 


o.oi 
o.oi 


0.01 

O.OI 
O.OI 

O.OI 
O.OI 
O.OI 
O.OI 

O.OI 


Marked  hemolysis  (7s  per  cent) 
Marked  hemolysis  (less  than  tube  i) 
Marked  hemolysis  (less  than  tube  i) 
Moderate  (less  than  2  or  3) 

Complete 
Complete 
Complete 
Almost  complete 

Moderate 
Moderate 
Moderate 
Moderate 

Marked  hemolysis  (75  per  cent) 


Almost  complete 
Complete 

o  (solution  clear) 


The  following  materials  were  used:  Sensitized  erythrocytes  from  goat,  washed 
in  5  per  cent  suspension,  i  day  old;  complement — fresh,  normal  guinea-pig  serum; 
antibody — inactivated  serum  of  guinea-pig  2,537,  recently  recovered  from  spotted 
fever;  antigen — «ggs  of  D.  modestus  (56  [57],  11,  F.  4),  10  days  old,  rich  in  bacilli. 

The  goat  corpuscles  were  sensitized  by  adding  o .  8  c.c.  of  immune  rabbit  serum  to 
20  c.c.  of  a  s  per  cent  suspension,  incubating  20  minutes  and  washing  once.  This  was 
done  in  order  to  avoid  the  necessity  of  making  separate  measurements  of  corpuscles 
and  of  the  hemolytic  serum. 

Antigen-|-antibody-|-complement  was  allowed  to  incubate  20  minutes  (i  hr.  in 
Experiment  3)  in  a  total  volume  of  0.3  c.c;  5  c.c.  suspension  of  corpuscles  was  then 
added  and  the  total  volume  raised  to  2  c.c.  by  the  addition  of  normal  salt  solution. 
The  mixture  thus  prepared  was  incubated  2  hours  and  then  placed  on  ice  over  night. 

were  negative.  In  a  fourth  experiment  dried  serum  of  an  immune 
horse  was  dissolved  and  used  as  an  antibody,  gi\dng  a  fairly  strong 
deviation  with  o.  2  c.c,  but  it  was  found  on  further  tests  that  0.4 
c.c.  of  this  serum  alone  would  bind  all  the  complement.  This  was 
possibly  due  to  the  presence  of  an  anti-complement  in  the  serum  of 


422  Contributions  to  Medical  Science 

the  horse,  it  ha\dng  been  actively  immunized  by  the  injection  of 
virulent  guinea-pig  blood. 

We  have  repeated  these  experiments  with  tick  eggs,  crushing 
a  given  number  of  them  (loo)  in  sterile  salt  solution  and  then  using 
decreasing  dilutions  of  this  emulsion.  Three  series  of  eggs  were  used : 
(i)  eggs  from  infected  ticks  (showing  large  numbers  of  organisms); 
(2)  eggs  from  normal  ticks;  and  (3)  eggs  from  ticks  raised  on  im- 
mune pigs.  The  three  series,  however,  showed  complete  lysis.  It  is 
true  that  no  lysis  was  obtained  in  the  100  per  cent  emulsion,  but  it 
was  found  that  tubes  without  antibodies  would  give  the  same  result. 
In  these  experiments  the  antigen-antibody-complement  mixture 
was  permitted  to  incubate  as  long  as  3-3^  hours. 

All  the  results  so  far  obtained,  while  showing  some  deviation, 
were  not  as  clear-cut  as  might  be  expected  in  a  disease  like  spotted 
fever  in  which  the  serum  of  recovered  animals  possesses  such  a 
marked  protective  power.'  We  therefore  undertook  the  testing  of 
various  tissue  extracts  and  of  sera  taken  from  animals  during  fever, 
in  the  hope  of  finding  an  efficient  antigen.  The  inactivated  serum 
of  recovered  guinea-pigs  served  as  antibody.  An  ox-rabbit  hemo- 
lytic system  was  used,  the  corpuscles  being  first  sensitized,  and  the 
exact  dosage  of  complement  established  for  each  experiment.  In 
the  case  of  various  bacterial  suspensions  used  as  antigens,  a  stand- 
ardized loop  of  a  24-hour  agar  culture  was  always  made  up  in  i  c.c. 
of  normal  salt  solution. 

In  one  experiment  varying  amounts  of  a  watery  extract 
of  macerated  lymph  glands,  spleen,  and  testicle  of  a  guinea- 
pig  which  had  died  of  spotted  fever  were  used  as  antigen,  with 
varying  amounts  of  antibody  (0.025  to  o.  i  c.c),  but  there  was  no 
deviation.^  This  negative  result  led  us  to  investigate  whether 
the  antibody  was  thermostable  or  whether  it  had  been  destroyed 
by  the  heating  at  56°  C.  We  found  that  both  fresh  and  heated 
immune  sera  protected  equally  well.  Evidently  the  diflSiculty 
remained  in  the  antigens.     We  therefore  made  separate  extracts 

'  Ricketts  and  Gomez,  Jour.  Infect.  Dis.,  1908,  5,  p.  221.     See  page  343  of  this  book. 

» It  may  be  stated  here  that  the  most  marked  internal  changes  during  a  course  of  spotted  fever  in  the 
guinea-pig  occur  in  the  lymph  glands,  testicle,  and  spleen.  Large  numbers  of  bodies  similar  in  appear- 
ance to  the  bacillus  found  in  the  tick  eggs  can  be  seen  in  the  spleen,  lymph  glands,  and  liver  when  sections 
are  stained  with  Giemsa  (Dr.  Maria  B.  Maver).  The  organism  must  also  be  present,  of  course,  in  the  blood, 
which  is  highly  virulent,  but  it  has  not  been  seen  equally  well  here  in  stained  preparations. 


Complement  Deviation  in  Spotted  Fever  423 

from  the  macerated  material  of  infected  pigs,  using  serum,  lungs, 
liver,  spleen  and  lymph  glands,  kidneys,  muscles,  brain,  and  intes- 
tine. Varying  quantities  were  used  (o.i  c.c.  to  0.5  c.c),  with  a 
constant  amount  of  immune  guinea-pig  serum.  The  organ  ex- 
tracts were  not  hemolytic.  The  results  of  the  experiment  were 
again  negative.  In  connection  with  these  experiments  a  series 
was  made  with  a  suspension  of  the  culture  of  the  "Spaniard"  or- 
ganism^ as  antigen  and  the  serum  of  guinea-pigs  recovered  from 
infection  with  it  and  from  Rocky  Mountain  spotted  fever  as  anti- 
body. 

As  will  be  seen  from  Table  2  complete  binding  of  the  comple- 
ment was  obtained  with  both  antibodies.    While  this  was  to  be  ex- 

'  The  "Spaniard"  organism,  culturally,  morphologically,  and  immunologically,  seems  to  be  practically 
identical  with  a  stock  culture  of  B.  cholera  suis  obtained  from  the  Department  of  Bacteriology  of  the 
University  of  Chicago.  It  crept  into  the  spotted  fever  passage  virus  derived  from  the  patient  "Spaniard" 
and  gradually  replaced  the  spotted  fever  virus.  Whether  it  actually  outgrew  and  "smothered"  the  virus 
is  not  known.  The  "Spaniard"  patient  was  suffering  from  the  Idaho  spotted  fever — a  strain  which  on 
three  previous  occasions  it  had  been  impossible  to  keep  going  by  passage  through  the  guinea-pig — so  that 
the  adventitious  infection  may  not  have  been  responsible  for  the  "dying  out"  of  the  spotted  fever  virus. 
By  proper  attention  to  dosage,  the  infection  produced  in  the  guinea-pig  by  the  intraperitoneal  injection 
of  the  "Spaniard"  organism  can  be  made  to  parallel  very  closely  in  some  respects  a  case  of  spotted  fever. 
There  is  the  same  three  days'  incubation  period,  the  subsequent  high  fever,  and  death  in  about  lo  days. 
Post  mortem  the  enlarged,  hemorrhagic  lymph  glands  and  swollen  spleen  are  conspicuous.  The  gangrenous 
ears  and  scrotum,  the  cutaneous  eruptions,  and  the  hemorrhages  in  the  region  of  the  pampiniform  plexus 
so  common  in  spotted  fever  are  wanting,  while  a  fibrinous  exudate  in  the  peritoneal  cavity,  never  found 
in  the  latter  disease,  is  always  present.  In  non-fatal  cases  the  temperature  of  spotted  fever  pigs  returns 
to  normal  in  about  lo  days  and  there  remains;  in  non-fatal  infections  with  the  "Spaniard"  organism,  a  low 
fever  may  persist  for  20  days  to  a  month  with  occasional  exacerbations.  Cultures  from  all  spotted  fever 
cases  remain  sterile;  the  specific  bacilli  may  always  be  cultivated  from  the  heart's  blood,  peritoneal 
exudate,  and  spleen  of  pigs  dying  from  infection  with  the  "Spaniard"  organism.  Finally,  recovery  from 
spotted  fever  does  not  confer  immunity  to  the  "Spaniard"  organism,  though  a  high  grade  of  specific  im- 
munity is  produced.  Both  cause  a  "hemorrhagic  septicemia"  but  the  "Spaniard"  organism  is  much  less 
prone  to  produce  hemorrhages  than  is  the  spotted  fever  virus. 

It  is  a  striking  coincidence  that  in  three  diseases  produced  by  micro-organisms  which  apparently  can- 
not be  cultivated,  namely  yellow  fever.  Rocky  Mountain  spotted  fever,  and  hog  cholera,  and  the  first 
two  of  which  at  least  may  be  transmitted  by  insects,  cholera-suis-like  organisms  should  have  intruded 
themselves.  This  fact,  coupled  with  those  detailed  in  this  paper  on  the  comparative  fixation  tests,  sug- 
gests that  there  may  be  something  more  concerned  here  than  mere  coincidence.  There  may  be  an  actual 
relationship  of  some  kind  between  the  various  causal  organisms.  With  this  idea  in  mind  the  following 
preliminary  experiment  with  precipitins  is  of  interest: 

The  precipitogens  consisted  of  filtered,  24-hour  broth  cultures  of  B.  cholera  suis,  "Spaniard,"  Staph, 
aureus,  and  B.  typhosus;  the  precipitins  were  contained  in  the  unhealed  sera  of  spotted  fever  immune 
guinea-pigs  Nos.  2,658  and  2,381  and  of  "Spaniard"  immune  guinea-pigs  Nos.  2,342  and  2,669.  Precipi- 
tates formed  in  those  tubes  containing  the  cholera  suis  and  "Spaniard"  filtrates  with  the  spotted  fever  and 
"Spaniard"  immune  sera  alike  in  a  serum  dilution  of  1-20  and  not  at  all  in  the  staphylococcus  and  typhoid 
tubes.     Control  tubes  remained  clear. 

''    Sample  Protocols. 
Precipitogen     Precipitin      NaCl  Sol. 


o.s  c.c. 

0 

or  c.c. 

1.49  c.c. 

o.s  c.c. 

0 

I    c.c. 

1.4    c.c. 

o.s  c.c. 

0 

i.S    c.c. 

0 

0 

I    c.c. 

1.9    c.c. 

424 


Contributions  to  Medical  Science 


pected  in  the  "Spaniard-Spaniard"  combination,  the  ''Spaniard"- 
spotted  fever  result  was  rather  unexpected.  While  these  controls 
had  been  intended  principally  as  a  check  on  our  technic  by  the  ad- 
dition of  known  factors,  and  showed  that  our  method  was  correct,  the 
result  indicated  either  that  the  "Spaniard"  culture  was  a  specific 
antigen  for  Rocky  Mountain  spotted  fever,  or  stood  in  a  group  rela- 
tionship to  the  same  (we  knew  that  the  first  supposition  was  not 
true)  or  that  the  guinea-pigs  had  at  some  previous  time  been  infected 
with  the  "Spaniard"  bacillus  and  were  therefore  immune. 

TABLE  2. 
CoMPLEstENT  De\tation  wtth  "Spaniard"  Bacillus  and  Spotted  Fever  and  "Spaniard" 

Immune  Serum. 


Antee 

oores 

Sensitized 
Corpuscles 

Antigen 

Culture 

"Span."  VI 

Complement 

Sp.  Fever 

"Spaniard" 

Results 

2,517 

2,343 

o.S 

0.025 

0.05 

0.01 

No  hemolysis 

0 

5 

0.025 

0.1 

O.OI 

No  hemolysis 

o 

S 

0.02s 

0.25 

O.OI 

No  hemolysis 

o 

S 

0.025 

o.S 

O.OI 

No  hemolysis 

o 

S 

0.05 

0.2s 

0.01 

No  hemolysis 

o 

5 

0.1 

0.25 

O.OI 

No  hemolysis 

o 

S 

0.0 

0.5 

O.OI 

Complete 

o 

5 

0.1 

0 

O.OI 

Complete 

o 

5 

0.02S 

0.05 

0.01 

No  hemolysis 

0 

S 

0.02S 

0.1 

O.OI 

No  hemolysis 

o 

S 

0.02S 

0.2s 

O.OI 

No  hemolysis 

o 

S 

0.025 

o.S 

O.OI 

No  hemolysis 

o 

S 

0.0s 

0.2s 

O.OI 

No  hemolysis 

O.S 

O.I 

0.2S 

O.OI 

No  hemolysis 

Tubes  incubated  for  30"  and  corpuscles  then  added  and  tubes  made  up  to  2  c.c.  with  normal  salt 
solution. 

Incubated  i  hr.  30"  and  then  placed  at  room  temperature  over  night. 


This  experiment  was  repeated  using  the  same  antibodies  and 
fresh  suspensions  of  "Spaniard,"  typhoid  bacilli,  and  Staphylococcus 
aureus  as  antigens.  The  results  with  the  typhoid  bacilli  and 
staphylococci  were  absolutely  negative  but  there  was  deviation  as 
in  the  previous  experiment  with  the  "Spaniard"  organism. 

The  effect  of  large  doses  of  antibody  causing  an  increase  of  he- 
molysis, as  noted  in  the  Ricketts  experiments  already  quoted,  was 
observed. 

Certain  preliminary  experiments  indicated  that  the  bacillus  of 
hog  cholera,  when  used  as  an  antigen  with  Rocky  Mountain  spotted 
fever  and  "Spaniard"  immune  sera  as  the  antibodies,  would  give 


Complement  Deviation  in  Spotted  Fever 


425 


evidence  of  binding  the  complement.  A  quantitative  experiment 
was  performed  in  order  to  compare  this  binding  with  the  results 
obtained  using  the  "Spaniard"  culture  as  antigen.  These  results 
are  shown  in  Table  3.  As  will  be  seen  in  Series  A  (spotted  fever- 
"Spaniard"),  there  is  first  a  progressive  decrease  in  the  amount  of 

TABLE  3. 


Groups 


Experi- 
ment 12 
Sensitized 
Corpus- 
cles 


Antibodies 


Sp.  Fever 
2,381 


"Spaniard' 
2,342 


Antigens 


Span. 
VI 


B.  cholera 
suis 


Comple- 
ment 


Results 


Group  A 

I 

2 

3 

4 

S 

6 

7 

8 

Group  B 

9 

10 

II 

12 

13 

14 

IS 

i5 

Group  C 

17 

18 

19 

20 

21 

22 

23 

24 

Group  D 

2S 

26 

27 

28 

29 

30 

31 

32 


o-S 

o-S 
0-5 
o-S 
o-S 


o.S 
o.S 
o-S 
o.S 
o.S 
o.S 
o.S 
0.5 

o.S 
o.S 
0.5 
o.S 
o.S 
o.S 
o.S 
o.S 


0.005 

0.0075 

o.oi 

0.02 

0.03 

0.04 

0.0s 

0.1 

0.005 

0.007s 

0.01 

0.02 

0.03 

0.04 

0.05 

0.1 


0.005 

0.0075 

0.01 

0.02 

0.03 

0.04 

0.05 

0.1 

0.005 

0.0075 

0.01 

0.02 

0.03 

0.04 

0.0s 

0.1 


0.0s 

0.05 
0.0s 
0.05 
0.05 
0.05 
0.05 
0.05 


0.02 
0.02 
0.02 
0.02 
0.02 
0.02 
0.02 
0.02 

0.02 
0.02 
0.02 
0.02 
0.02 
0.02 
0.02 
0.02 

0.02 
0.02 
0.02 
0.02 
0.02 
0.02 


0.05 
0.0s 
0.05 
0.05 
0.05 
0.05 
0.05 
0.05 


Incomplete  laking 
Incomplete  laking 
Trace  of  laking 
Trace  of  laking 
Trace  of  laking 

o 
Slight  laking 
Moderate  laking 

Incomplete  laking 
Incomplete  laking 
Incomplete  laking 
Trace  of  laking 
Trace  of  laking 
Least  laking 
Slightly  more  laking 
Slightly  more  laking 

Incomplete  laking 
Slight  laking 
Slight  laking 

o 
Complete  laking 
Complete  laking 
Complete  laking 
Complete  laking 


Complete 
Complete 
Complete 
Complete 
Complete 
Complete 
Complete 
Complete 


laking 
laking 
laking 
laking 
laking 
laking 
laking 
laking 


laking  reaching  a  maximum  in  tube  6  in  which  we  find  complete 
binding  and  there  follows  increased  laking  in  tubes  7  and  8.  Series 
B  (spotted  fever+hog  cholera)  shows  a  similar  reaction,  though  the 
best  tube  still  shows  a  trace  of  laking.  In  Series  C  ("Spaniard"+ 
"Spaniard")  we  find  a  marked  deviation  in  the  first  four  tubes  (90 
per  cent  to  100  per  cent)  and  complete  laking  in  the  last  four  tubes. 
Series  D,  on  the  other  hand  ("Spamard"-hog  cholera),  gave  com- 
plete laking.  This  experiment  was  repeated  twice,  using  sera  from 
different  immune  pigs,  and  similar  results  were  obtained  with  the 


426  Contributions  to  Medical  Science 

exception  that  now  the  "Spamard"-hog  cholera  combination  gave 
the  reverse  of  the  result  in  Experiment  4,  in  which  we  found  a  nega- 
tive reaction.  Further  experiments  have  confirmed  these  results, 
and  also  seem  to  show  (i)  that  the  serum  of  normal  guinea-pigs 
does  not  contain  the  specific  antibody;  (2)  that  the  spotted  fever 
immune  serum  causes  no  deviation  in  the  presence  of  typhoid, 
anthrax,  dysentery  (Shiga),  and  Friedlander  bacilli,  staphylococci, 
and  cholera  germs,  although  it  has  a  specific  afl^ty  for  certain 
strains  of  bacteria  of  the  hog  cholera  group.  The  spotted  fever  an- 
tibody, in  certain  proportions,  will  bind  complement  with  the  hog 
cholera  and  "Spaniard"  bacilH,  and  the  "Spaniard"  immune  serum 
will  bind  with  the  same  bacilli.  We  determined,  however,  that  pigs 
immune  to  Rocky  Mountain  spotted  fever  are  not  protected  against 
the  "Spaniard"  organism. 

We  have  failed  to  find  a  parallelism  between  the  bacteriolytic 
power  of  these  immune  sera  on  hog  cholera  bacilH  and  the  "Span- 
iard" bacillus,  as  determined  by  the  Neisser-Wechsberg  technic, 
and  their  power  to  deviate  complement.  That  is,  the  bactericidal 
power  of  normal  sera  and  of  "Spaniard"  immune  sera  was  almost 
absolutely  identical,  while  only  the  immune  sera  supplied  the  am- 
boceptor necessary  for  the  deviation  of  the  complement.  Appar- 
ently the  presence  of  substances  in  sera  which  cause  a  fixation  or 
deviation  of  complement  need  not  imply  the  presence  of  bacterio- 
lytic or  protective  antibodies.  A  somewhat  similar  phenomenon 
has  been  observed  by  Torrey  and  others.^ 

conclusions. 

Positive  deviation  of  complement,  using  spotted  fever  antigen 
and  antibody,  has  been  obtained  in  but  one  instance,  and  the  results 
of  experiments  undertaken  to  confirm  this  positive  finding  have 
been  negative. 

The  negative  results  are  probably  to  be  explained  by  the  small 
quantities  of  organisms  present  in  any  one  preparation  of  antigen 
and  by  the  admixture  of  various  substances  in  the  antigens  which 
in  themselves  absorb  or  neutralize  complement,  this  in  turn  pro- 

■  Torrey,  Jour.  Med.  Res.,  1910,  22,  p.  95. 


Complement  Deviation  in  Spotted  Fever  427 

hibiting  the  use  of  the  large  doses  of  antigen  which  might  otherwise 
be  effective. 

The  protective  power  of  spotted  fever  immune  serum  is  not 
destroyed  by  heating  to  56°  C.  for  30  minutes. 

An  apparent  reactivation  of  heated  serum  (antibody)  was  met 
with  in  many  cases  which,  by  causing  hemolysis,  tended  to  mask 
results.     This  might  become  a  source  of  error  in  work  of  this  kind. 

An  organism  probably  identical  with  the  hog  cholera  bacillus 
replaced  the  spotted  fever  virus  in  one  of  the  passages.  The  serum 
of  guinea-pigs  immune  to  spotted  fever  and  of  guinea-pigs  immune 
to  this  organism  deviated  the  complement  alike  when  a  suspension 
of  the  above  bacterium  was  used  as  antigen.  This  fact,  together 
with  other  considerations  mentioned  in  this  paper,  suggests  that 
there  may  be  a  definite  relationship  of  some  kind  between  the 
virus  of  Rocky  Mountain  spotted  fever  and  the  hog  cholera 
bacillus. 

Our  results  suggest,  further,  that  the  presence  of  substances 
in  sera  which  cause  a  complement-fixation  need  not  imply  the 
presence  of  bactericidal  or  protective  antibodies. 


TIME   RELATIONSHIPS   OF   THE  WOOD-TICK   IN   THE 

TRANSMISSION  OF  ROCKY  MOUNTAIN 

SPOTTED   FEVER/ 

JosiAH  J.   Moore. 

{From  the  Pathological  Laboratory  of  the  University  oj  Chicago.) 

The  chief  object  of  the  following  experiments,  undertaken  at 
the  suggestion  of  the  late  Dr.  Howard  T.  Ricketts,  was  to  determine 
(i)  the  minimum  duration  of  feeding  by  infected  tick  necessary  to 
infect  the  guinea-pig;  (2)  the  minimum  duration  required  for  the 
infection  of  the  tick  from  the  infected  guinea-pig;  and  (3)  the 
length  of  the  incubation  period  in  the  tick,  that  is,  the  period  of 
time  required  for  the  etiological  factor  to  establish  itself  in  the 
tick  and  render  it  infective. 

Duration  of  feeding  by  infected  tick  necessary  to  infect  the  guinea- 
pig. — All  the  ticks  used  in  this  experiment  were  bred  in  the  labora- 
tory in  tick-proof  cages.  They  were  of  two  species,  Dermacentor 
venustus  and  Dermacentor  modestus.  The  parents  of  the  former 
were  the  second  generation  of  that  species  raised  in  the  laboratory, 
the  original  ancestors  having  been  procured  from  the  Bitter  Root 
Valley  in  Montana  in  the  spring  of  1907.  These  had  been  infected 
in  the  usual  manner  with  the  Montana  strain  of  fever  and  had  trans- 
mitted this  from  generation  to  generation  up  to  the  present  time. 
The  parents  of  the  latter  species  (Dermacentor  modestus)  were 
collected  in  Idaho  in  the  spring  of  1909  and  these  also  were  infected 
with  the  Montana  strain. 

The  method  was  as  follows:  In  the  group  of  experiments  in 
Table  i ,  the  tick  was  first  permitted  to  feed  for  five  or  ten  days  on 
a  normal  guinea-pig  to  test  its  infectivity.  After  making  sure  that 
it  was  infected,  the  tick  was  removed  and  kept  at  room  temperature 
for  a  variable  period ;  then  fed  on  a  normal  guinea-pig  for  a  certain 
number  of  hours;  again  removed  and  kept  at  room  temperature. 
The  feedings  were  repeated  at  variable  intervals  on  new  guinea- 
pigs  until  the  tick  died. 

'From  Jour.  Inject.  Dis.,  1911,  8,  p.  339. 

428 


The  Wood-Tick  in  Transmission  of  Spotted  Fever    429 

In  the  second  group  of  experiments  (Table  2)  the  ticks  were  not 
given  a  preliminary  test,  but  the  first  feed  was  regarded  as  a  time 
test;  in  all  other  respects  the  experiments  were  similar. 

When  testing  or  feeding  an  individual  tick,  the  guinea-pig  was 
placed  in  a  pillory  and  the  tick  allowed  to  attach  itself  to  the  ear. 
The  time  of  feeding  was  taken  from  the  minute  the  tick  became  so 
firmly  attached  that  when  the  tick  body  was  flipped  back  it  could 
not  be  detached  from  the  pig. 

In  the  beginning  of  the  work  it  was  thought  that  a  feeding  of 
one  hour  would  be  sufficient  to  infect  a  guinea-pig,  but  it  was  soon 
found  necessary  to  extend  this  period  to  five  hours.  If  the  tick 
proved  infective  in  the  five-hour  test  the  time  could  be  reduced 
in  the  second  test,  or  if  the  infection  were  not  transmitted  to  the 
guinea-pig  in  the  first  test,  the  time  could  be  lengthened. 

The  results  of  the  experiments  as  shown  by  the  tabular  summary 
of  Tables  i  and  2  are  not  constant.  However,  when  it  is  consid- 
ered that  the  tick  is  a  living  factor,  this  inconstancy  is  partially 
explained.  The  tick  is  a  slow  feeder.  The  hungriest  and  most 
vigorous  require  from  15  to  20  minutes  to  attach  themselves  firmly 
by  means  of  the  palpi,  and  without  this  firm  attachment  feeding 
is  impossible.  In  other  ticks  the  process  is  much  slower,  and  after 
two  or  three  days  of  attachment  there  is  Httle  evidence  of  the 
ingestion  of  blood. 

Table  i  shows  that  the  minimum  duration  of  tick  feeding  neces- 
sary to  infect  a  guinea-pig  was  found  to  be  one  hour  and  forty- 
five  minutes  (Experiment  I,  with  Dermacentor  modestus.  Female 
90).  In  Experiment  XV,  D.  modestus,  Female  no,  was  infected  in 
a  two-hours'  feed  and  again  in  a  three-hours'  feed.  A  five-hours' 
feed  inoculated  in  Experiments  IV,  V,  XIII,  XV,  XVI,  and  XVIII; 
an  eight-hours'  in  Experiment  XII;  a  ten-hours'  in  Experiments  I, 
XI,  and  XIV;  a  fifteen-hours'  in  Experiments  XV  (twice),  XVIII, 
and  XIX;  a  twenty-hours'  in  Experiments  III,  X,  XIV,  XVII, 
and  XX.  Twenty-seven  five-hour  tests  were  tried,  and  six,  or  22  per 
cent,  infected;  ten  ten-hour  tests,  of  which  three,  or  30  per  cent, 
infected;  eight  fifteen-hour  tests,  in  which  four,  or  50  per  cent,  were 
positive;  six  twenty-hour  tests,  with  five,  or  83  per  cent,  positive; 
nine  one-  to  three-hour  tests  and  three,  or  33  per  cent,  infected.   To 


430 


Contributions  to  Medical  Science 


TABLE  I. 

DxjaATiON  OF  Tick-Feeding  Required  for  the  Infection  of  the  Gcinea-Pig. 

Ticks  previously  tested  and  proved  infective. 


Experiment 

Tick  Species 
and  Number 

End  of 

Infectivity 

Test 

Interval 

Length  of 

First  Test 

and 

Date 

1 
Pi 

> 
C 

Length  of 

Second 

Test 

and  Date 

■3 

(2 

1 

V 

a 

I        

D.  modestus 
F.  90 

D.  venustus 
M.  196 

D.  modestus 
M.  100 

D.  modestus 

F.  87 
D.  modestus 

F.  88 
D.  venustus 

F.  198 
D.  venustus 

F.  199 
D.  venustus 

F.  202 
D.  venustus 

F.  203 
D.  venustus 

M.  204 
D.  modestus 

F.  103 
D.  modestus 

F.  108 
D.  modestus 

F.  106 
D.  modestus 

F.  107 
D.  modestus 

F.  no 
D.  modestus 

F.  los 

9/1 S 
9/1 S 

10/5 

9/21 
9/20 
10/2S 
10/25 
11/2 
11/16 
11/19 
10/18 
11/28 
11/22 
11/28 
11/29 
ri/22 

3  days 

7  days 
17  days 

43  days 

44  days 
9  days 

9-10  days 
10  days 
14  days 
13  days 
37  days 
10  days 
25  days 
19  days 
21  days 
31  days 

18  hr. 
9/18 

1  hr. 

9/22 

10  hr. 
10/22 

Shr. 
11/3 

shr. 
11/3 

Shr. 
11/4 

Shr. 
11/4 

shr. 
11/12 

shr. 
11/30  , 

shr. 
12/2 

shr. 
12/8 

shr. 
12/8 

shr. 
12/17 

IS  hr. 
12/17 

IS  hr. 
12/20 

3-S  hr. 
12/23 

+ 

+ 
+ 

+ 
+ 

4  days 

4  days 
4  days 

8  days 

8  days 

26  days 

18  days 

20  days 

14  days 

21  days 

17  days 

tick  dead 
12/12 
23  days 

20  days 

21  days 

15  days 

10  br. 
9/22 

Shr. 

9/25 

shr. 
10/26 

2jhr. 
ii/ii 

1  hr. 
11/11 

Shr. 
1 2/1 

shr. 
ir/22 

shr. 
12/2 

IS  hr. 
12/14 

20  hr. 
12/23 

10  hr. 
12/19 

8hr. 
1/9 

20  hr.  disc 
ed  i/io 
io(?)  hr.di 
ded  i/ii 
Shr. 
1/7 

+ 

+ 
+ 

ard- 
scar- 

16  days 

n 

tick  dead 

rrr 

9/27 
13  days 

IV 

19  days 

V 

19  days 

VI 

12  days 

vn 

20  days 

yrrr        

tick  dead 

rx 

12/S 
tick  dead 

X     

12/26  ; 
IS  days 

XI 

tick  dead 

xn 

2/23 

xm        

19  days 

XIV 

16  days 

XV 

20  days 

XVI 

23  days 

TABLE  2. 

Duration  of  Tick-Feeding  Required  for  the  Infection  of  the  Guinea-Pig. 

Ticks  not  previously  tested  for  infectivity. 


Experiment 

Tick  Species 
and  Number 

Date  of 
Moulting 

Interval 

Duration  of 
First  Test 
and  Date 

Result 

Interval 

xvn 

xvm 

D.  modestus 

F.  128 
D.  venustus 

M.  222 
D.  modestus 

M.  129 
D.  modestus 

M.  130 

Unknown 
Unknown 
Unknown 
Unknown 

Unknown 
Unknown 
Unknown 
Unknown 

Shr. 
1/17 

iShr. 
1/18 

shr. 
1/19, 

shr. 
1/19 

- 

18  days 
10  days 

XIX 

21  days 

XX 

23  days 

The  Wood-Tick  in  Transmission  of  Spotted  Fever    431 


TABLE  I. 

Duration  of  Tick-Feeding  Required  for  the  Infection  of  the  Guinea-Pig. 

Ticks  previously  tested  and  proved  infective. 


^ 

Length 

Length 

Length 

Length  of 

of 

of 

Third 

Fourth 

■3 

Fifth 

Sixth 

•5  oQ 
p,  *>  "^ 

Test  and 

•3 

t 

Test 

3 

> 

Test 

3 

> 

Test 

•g 

r, 

3 

Date 

S 

4> 

and 

8 

and 

and 

? 

2 

^ 

►s 

Date 

P< 

a 

Date 

^ 

A 

Date 

P< 

5 

^a>ca 

^ 

I  hr.  45  m. 

+ 

11  days 

6  days 

+ 

tick  dead 

10/9 

10/19- 

10/25 



10/30 

10  hr. 

_ 

22  days 

II  days 

+ 

28  days 

20  hr. 

+ 

10  days 

10  hr. 

_ 

11/9 

12/1- 
12/12 

1/9 

10/19 

2ihr. 

— 

tick  dead 

"/30 

1 2/5 

2jhr. 

— 

tick  dead 

"/30 

12/s 

10  hr. 

— 

tick  dead 

12/13 

12/16 

10  hr. 

— 

tick  dead 

12/13 

12/18 

Shr. 

20  days 

20  hr. 

+ 

II  days 

10  hr. 

14  days 

10  hr. 

1/7 

1/27 

2/7 

2/21 

Shr. 

li  days 

Shr. 

tick  dead 

1/28 

2/9 

2/20 

20  hr. 

+ 

II  days 

10  hr. 

+ 

tick  dead 

1/27 

2/7 

2/1S 

IS  hr. 

+ 

8  days 

Shr. 

+ 

14  days 

3hr. 

+ 

9  days 

2  hr. 

+ 

3  day 

s     I  hr. 

— 

2/1 

2/9 

2/23 

3/4 

3/7 

Shr. 

+ 

tick  dead 

2/1 

2/10 

TABLE  2. 

Duration  of  Tick-Feeding  Required  for  the  Infection  of  the  Guinea-Pig. 

Ticks  not  previously  tested  for  infectivity. 


Duration  of 

Second  Test 

and  Date 

Result 

Interval 

Duration  of 
Third  Test 
and  Date 

Result 

Interval 

Duration  of 

Fourth  Test 

and  Date 

Result 

Shr. 

^/'*    I. 
IS  hr. 

1/28 

IS  hr. 

2/9 

+ 
+ 
+ 

12  days 

15  days 

16  days 
tick  dead 

15  hr. 

2/16 

5  hr. 
2/1 1 

10  (?)hr. 
2/25 

+ 

20  days 
12  days 
tick  lost 

20  hr. 

3hr. 
2/23 

+ 

2/11 

432  Contributions  to  Medical  Science 

account  for  the  higher  percentage  of  infections  in  the  one-  to  three- 
hour  than  in  the  five-  and  ten-hour  tests  we  find  that  in  the  former 
cases  two  highly  infected  and  voracious  ticks  caused  the  three 
inoculations,  while  in  the  latter  the  ticks  were  apparently  not  so 
heavily  infected,  and  were  only  average  feeders. 

From  the  data  given  it  is  observed  that  the  average  time  for 
infecting  the  guinea-pig  by  the  wood-tick  is  about  ten  hours,  and 
that  twenty  hours  is  almost  surely  infective.  Only  once  did  this 
fail,  but,  on  repeating  (Experiment  X),  a  positive  result  was  obtained. 

D.  modestus.  Female  90  and  Female  no,  gave  the  best  results, 
infecting  the  animals  upon  which  they  were  tested  at  practically 
each  feed.  In  Experiment  XIII  the  first  five-hours'  feed  was 
positive,  while  two  others  repeated  later  at  varying  intervals  did 
not  cause  infection.  The  results  obtained  in  this  experiment,  in 
Experiment  XVI,  and  also  in  Experiment  XVII,  where,  after  a 
negative  fifteen-hours'  test,  there  followed  positive  twenty-  and  five- 
hour  tests,  may  have  been  due  to  disincHnation  of  the  tick  to  feed 
vigorously.  In  Experiments  I  and  III  the  weakened  condition  of 
the  ticks  necessitated  long  feeds  to  restore  their  vitaHty. 

From  the  tabulated  summary  it  appears  that  both  males  and 
females  of  the  modestus  species  gave  more  positive  results  than 
those  of  the  venustus.  This  was  probably  due  to  the  fact  that 
the  former  were  younger  and  more  vigorous  and  therefore  fed  more 
rapidly. 

Duratioft  of  feeding  required  for  the  infection  of  the  tick  and  length 
of  incubation  period  in  the  tick. — The  technic  in  this  series  of  experi- 
ments was  as  follows:  A  number  of  normal  ticks  were  allowed  to 
feed  on  an  infected  guinea-pig  for  a  definite  number  of  hours;  they 
were  then  removed  and  kept  at  room  temperature  for  several  days, 
when  feeding  was  again  permitted,  this  time,  however,  on  a  normal 
guinea-pig,  for  a  definite  period.  Both  species,  D.  venustus  and  D. 
modestus,  were  tested.  The  infected  guinea-pigs  on  which  the  ticks 
were  fed  were  regular  passage  animals,  and  were  used  between  their 
second  and  fifth  days  of  fever. 

In  only  two  of  the  27  was  the  spotted  fever  virus  demonstrated 
in  the  tick,  so  that  the  results  cannot  be  considered  conclusive. 
It  seems  highly  probable  that  a  much  shorter  feed  is  required  for 


The  Wood-Tick  in  Transmission  of  Spotted  Fever    433 


TABLE  3. 
Duration  of  Feeding  Necessary  to  Infect  Tick  and  Incctbation  Period  in  the  Tick  . 


Exp. 

Tick  Species 

Date  of 

Length 

of 
Feeding 

Inter- 

Date 

Length 

Re- 

Inter- 

Date of 

Second 

Test 

Length 

Re- 

and Number 

Feeding 

val 

of  Test 

of  Test 

sult 

val 

of  Test 

sult 

I 

D.  venustus 

M.  204 

iVs 

S  hrs. 

7  days 

12/12 

10  hrs. 

— 

M.  205 

12/5 

5  hrs. 

14  days 

12/19 

10  hrs. 

— 

F.  206 

12/s 

5  hrs. 

tick  died 

before  b 

eing  test 

ed 

M.  207 

12/3 

10  hrs. 

7  days 

12/12 

5  hrs. 

— 

M.  208 

12/S 

10  hrs. 

13  days 

12/18 

20  hrs. 

— 

F.  209 

12/s 

10  hrs. 

39  days 

1/13 

IS  hrs. 

— 

M.  210 

12/S-6 

20  hrs. 

6  days 

12/12 

20  hrs. 

— 

29  days 

2/2 

IS  hrs. 

— 

M.  211 

12/S-6 

20  hrs. 

13  days 

12/18 

20  hrs. 

— 

F.   212 

12/5-6 

20  hrs. 

39  days 

1/13 

15  hrs. 

— 

II 

D.  venustus 

M.  214 

1/9 

10  hrs. 

tick  died 

before  b 

eing  test 

ed 

M.  215 

1/9 

10  hrs. 

4j  days 

1/14 

20  hrs. 

— 

M.  216 

1/9 

10  hrs. 

25  days 

2/4 

20  hrs. 

— 

F.   217 

1/9-IO 

20  hrs. 

4  days 

1/14 

20  hrs. 

— 

F.   218 

1/9-10 

20  hrs. 

23  days 

1/31 

15  hrs. 

— 

M.  219 

1/9-10 

20  hrs. 

41  days 

2/18 

15  hrs. 

— 

F.   220 

1/9-10 

25  hrs. 

4  days 

1/14 

20  hrs. 

— 

40  days 

2/18-3/4 

14  days 

+ 

M.  221 

1/9-10 

25  hrs. 

24  days 

2/2 

IS  hrs. 

— 

Ill 

D.  venustus 

M.  22s 

1/24 

25  hrs. 

37  days 

3/2 

20  hrs. 

— 

M.  226 

1/24 

25  hrs. 

tick  died 

before  b 

eing  test 

ed 

IV 

D.  modestus 

M.  168 

3/1-2 

25  hrs. 

20  days 

3/22 

20  hrs. 

— 

M.  173 

3/1-2 

40  hrs. 

10  days 

3/12 

20  hrs. 

— 

M.  174 

3/1-2 

40  hrs. 

20  days 

3/22 

20  hrs. 

— 

V 

D.  venustus 

F.  232 

3/24-25 

25  hrs. 

35  days 

4/28 

25  hrs. 

— 

VI 

D.  modestus 

F.  178 

3/24-30 

25  hrs. 

30  days 

4/28 

30  hrs. 

+ 

F.  179 

3/29-30 

25  hrs. 

38  days 

5/6 

25  hrs. 

infection  and  that  the  incubation  period  in  the  tick  is  shorter  than 
is  indicated  by  these  experiments.  The  complete  protocols  of 
the  two  tests  in  which  complete  results,  as  indicated  by  the  trans- 
mission of  the  disease  by  the  tick  to  the  normal  guinea-pig,  were 
obtained,  follow. 

D.  venustus,  Female  220,  was  attached  to  infected  guinea-pig  2,547  for  25  hours 
on  January  9-10.  No  hyperemic  area  was  observed  around  the  bite  and  no  feces  were 
passed.  On  January  14,  after  an  interval  of  four  days,  it  attached  itself  to  normal 
guinea-pig  2,713  and  was  removed  after  20  hours.  The  tick  did  not  appear  to  feed 
much,  not  increasing  any  in  size,  and  passing  no  feces.  The  temperature  of  guinea-pig 
2,713  was  taken  for  two  weeks  but  did  not  rise  above  103.4°  F.  A  month  later,  Feb- 
ruary 27,  the  pig  was  given  an  immunity  test  of  i  c.c.  of  blood  from  an  infected  pig. 
It  became  infected  and  died,  the  organs  showing  changes  t>T)ical  of  spotted  fever. 
This  proved  conclusively  that  the  guinea-pig  had  not  been  infected  by  the  tick,  since 
Ricketts  and  Gomez"  have  shown  that  one  attack  of  spotted  fever  renders  the  guinea- 
pig  immune  to  a  second  inoculation. 

On  February  18  Female  220  attached  itself  to  normal  guinea-pig  2,779.  It  was 
found  ofif  March  5,  having  been  attached  for  15  days.  It  had  increased  markedly  in 
size  during  the  feeding.  A  hyperemic  area  about  2  cm.  in  diameter  was  observed 
around  the  bite. 

*Jour.  Infect.  Dis.,  1908,  s,  p.  221. 


434  Contributions  to  Medical  Science 

TEMPERATURE  OF  GUINEA-PIG  2,779- 

February  18 103. 6°  F.  February  25 102. 6°  F.  March    4 105. 2*  F. 

February  19 104°      F.  February  26 102 . 4°  F.  March    s 105°      F. 

February  20 103. 6°  F.  February  27 March    6 F. 

February  21 103. 8°  F.  February  28 102. 6°  F.  March    7 ios.6°F. 

February  22 103. 6°F.  March        i 102. 4°F.  March    8 105. 2°F. 

February  23 102 . 9°  F.  March        2 March    9 104°     F. 

February  24 102. 6°F.  March        3 105°  F.  March  10 ioi.2"'F.* 

March  11 Dead. 

•  Blood  taken  from  heart. 

Autopsy. — Axillary  glands  enlarged  and  congested.  Spleen  twice  the  normal 
size  and  of  a  deep,  bluish-red  color.  The  heart,  lungs,  and  intestines  normal.  The 
suprarenal  glands  and  liver  slightly  enlarged  and  congested.  The  external  genitals 
swollen  and  the  skin  slightly  hemorrhagic. 

Diagnosis. — Spotted  Fever.  Blood  drawn  from  the  heart  on  March  10  was  in- 
oculated into  guinea-pig  2,831,  which  ran  a  typical  spotted-fever  temperature.  The 
scrotum  became  swollen,  the  ears  became  necrotic  and  finally  sloughed  off.  The 
guinea-pig  recovered  and  later  was  given  an  immunity  test  during  which  its  temperature 
remained  normal.  All  these  facts  prove  beyond  doubt  that  guinea-pig  2,779  had 
spotted  fever. 

In  most  instances  it  has  been  found  that  an  infected  tick  will 
produce  the  fever  in  a  guinea-pig  after  an  incubation  period  in  the 
pig  of  five  or  six  days.  In  the  present  case  this  is  doubled.  Two 
assumptions  may  explain  the  results:  (i)  either  the  tick  did  not 
feed  for  several  days  after  becoming  attached  to  guinea-pig  2,779; 
or  (2)  the  amount  of  virus  ingested  by  the  tick  in  its  feed  upon  in- 
fected guinea-pig  2,547  may  have  been  exceedingly  small,  since  the 
tick  did  not  feed  actively,  as  is  shown  by  the  absence  of  a  hyperemic 
area  around  the  bite,  and  this  small  quantity  of  virus  may  not  have 
increased  in  the  body  of  the  tick  sufficiently  to  infect  a  guinea-pig 
until  the  tick  had  enjoyed  for  some  days  what  we  may  regard  as 
a  very  favorable  condition  of  attachment  to  a  guinea-pig. 

On  March  29  D.  modestus,  Female  178,  attached  itself  to  the  ear  of  infected 
guinea-pig  2,771,  where  it  was  allowed  to  remain  for  25  hours.  Considerable  feces 
were  passed  by  the  tick,  and  a  slight  hemorrhagic  area  surrounded  the  bite.  After 
an  intermission  of  30  days,  during  which  the  tick  was  transferred  from  Chicago  to 
Montana,  it  attached  itself  on  April  28  to  normal  guinea-pig  3,004,  being  removed 
after  30  hours.     On  May  5  the  temperature  of  the  guinea-pig  rose  to  104°  F. 

TEMPERATURE  OF  GUINEA-PIG  3,004. 

April  28 103°      F.  May    5 104°      F. 

April  29 102. 6°  F.  May    6 105. 1'F. 

April  30 102. 8°  F.  May    7 106°      F. 

May    1 103°      F.  May    8 106. 2°  F. 

May    3 103 . 2°  F.  May    9 105°      F. 

May    3 103. 1'F.  May  10 104.1°  F.  scrotum  swollen. 

May    4 103 . 6°  F.  May  11 103°      F.  scrotum  hemorrhagic. 

May  12 103 . 4°  F.     Recovered. 


The  Wood-Tick  in  Transmission  of  Spotted  Fever    435 

An  immunity  test  was  given  May  16,  but  no  fever  resulted,  showing  that  guinea- 
pig  3,004  had  been  infected  the  first  time.  The  incubation  period  in  the  tick  in  this 
case  was  30  days  with  a  minimum  duration  for  infecting  the  tick  of  25  hours.  With 
D.  modestus.  Female  179,  which  was  attached  the  same  length  of  time  as  Female  178 
on  guinea-pig  2,771,  but  was  given  a  25-hour  test  after  an  interval  of  38  days,  a  nega- 
tive result  was  obtained. 

In  previous  experiments  the  duration  of  feeding  required  for 
the  infection  of  the  tick  was  shown  to  be  less  than  was  found  here, 
20  hours  being  the  shortest  period.^  The  incubation  period  is, 
perhaps,  "represented  only  by  the  time  required  for  the  distribution 
of  the  virulent  organisms  throughout  the  tick's  body  and  eventu- 
ally into  its  salivary  glands.  In  a  number  of  instances  the  tick 
proved  virulent  immediately  following  its  removal  from  the  in- 
fected animal"  (Ricketts).^  In  the  latter  cases,  however,  the  tick 
was  permitted  to  feed  for  several  days  on  the  infected  guinea-pig 
and  thus  the  incubation  period  may  have  been  reduced  by  the 
tick's  remaining  attached  to  the  guinea-pig,  or  it  may  be  that  the 
chances  that  the  tick  will  infect  the  susceptible  guinea-pig  are 
greatest  immediately  after  its  proboscis  has  been  contaminated  by 
feeding  on  an  infected  animal. 

More  experiments  will  have  to  be  performed  before  any  definite 
statements  can  be  made  relative  to  the  length  of  the  incubation 
period  in  the  tick.  The  great  number  of  negative  results  may  have 
been  due  to  the  fact  that  the  ticks  were  raised  under  artificial  con- 
ditions and  did  not  feed  as  rapidly  as  those  obtained  from  nature. 
It  will  probably  be  found  that  ticks  taken  directly  from  the  woods 
will  give  better  results.  Similar  experiments  with  fresh  ticks  were 
to  be  undertaken  in  the  field  during  the  spring  of  1910,  but  the 
unfortunate  death  of  Dr.  Ricketts  prevented  the  carrying-out  of 
the  work.  It  will,  however,  probably  be  completed  at  some  future 
date. 

SUMMARY. 

The  minimum  duration  of  feeding  necessary  for  a  tick  to  infect 
a  guinea-pig  was  found  to  be  one  hour  and  forty-five  minutes.  The 
average  time  necessary  seems  to  be  about  ten  hours,  while  twenty 

'Ricketts,  H.  T.,  Jour.  Am.  Med.  Assn.,  1907,  49,  p.  24. 
'Med.  Record,  1909,  76,  p.  843;  page  383  of  this  book. 


436  Contributions  to  Medical  Science 

hours  were  almost  constantly  infective.  The  duration  of  feeding 
necessary  to  infect  a  tick  is  approximately  twenty-five  hours, 
while  the  minimum  incubation  period  in  the  tick  was  not  definitely 
determined.  With  ticks  obtained  from  nature  it  is  possible  that 
the  duration  of  feeding  necessary  to  infect  the  tick  and  the  incu- 
bation period  in  the  tick  will  be  found  to  be  much  less  than  is  indi- 
cated here. 


TRANSMISSION  OF   SPOTTED   FEVER  BY  THE  TICK 

IN  NATURE.^' ^ 

Maria  B.   Maver. 

(From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

The  discovery  of  ticks  in  nature  carrying  the  virus  of  spotted 
fever  is  published  by  Dr.  Ricketts^  (page  312),  who  points  out  the 
importance  of  this  fact  in  connection  with  the  theory  of  transmission 
of  spotted  fever  by  the  tick. 

The  following  experiments  were  made  in  order  to  throw  further 
light  on  the  natural  infectivity  of  the  tick. 

Six  hundred  and  fifty-six  ticks  were  collected  during  the  spring 
months  of  1909:  254  of  the  species  D.  modes tus  and  402  D.  venus- 
tus,  for  the  purpose  of  studying  the  infectivity  of  the  tick  in  nature. 
These  ticks  were  found  on  cows,  bushes,  and  vegetation  in  the  Lo 
Lo  Valley  and  Owl  Canon,  Montana,  and  in  the  vicinity  of  Poca- 
tello,  Idaho. 

Fourteen  groups  were  arranged,  consisting  of  25  male  and  25 
female  ticks  each.  Each  group  was  placed  in  a  new  tick-proof 
sack,  with  a  selected  guinea-pig  of  medium  size;  nine  sacks  of  Mon- 
tana ticks  (D.  venustus)  and  five  sacks  of  Idaho  ticks  (D.  modestus) 
were  arranged  in  this  way.  The  daily  temperature  of  each  pig 
was  taken  and  the  number  of  ticks  attached  noted. 

In  Groups  IV  and  V  the  guinea-pig  became  infected.  The 
ticks  in  both  groups  were  gathered  in  the  Lo  Lo  Valley  from  cows. 

Experiment  IV. — Normal  guinea-pig  2,155  was  the  host  of 
Group  IV.  For  two  weeks  this  pig  kept  up  a  vigorous  scratching. 
The  ticks  were  gathered  daily  and  placed  among  the  hair.  Many 
were  attached  on  the  fourth  day.  On  the  sixth  day  two  engorged 
females  were  found.  On  the  14th  day  the  i&rst  indication  of  a  disease 
was  noted  in  a  temperature  of  104°,  followed  on  the  next  two  days 
by  a  rise  to  106°,  with  hemorrhagic  vulva;  i|  c.c.  of  blood  was  with- 

'  From  Jour.  Infect.  Dis.,  1911,  8,  p.  322. 

'This  work  was  supported  by   a  grant  from  the  Memorial  Institute  for  Infectious  Diseases, 
Chicago. 

J  Jour.  Am.  Med.  Assn.,  1907,  49,  p.  24. 

437 


438  Contributions  to  Medical  Science 

drawn  from  the  heart,  and  the  pig  died  two  days  later,  with  lesions 
characteristic  of  spotted  fever. 

The  heart's  blood  taken  from  the  pig  was  immediately  injected 
into  normal  guinea-pig  2,203,  producing  a  typical  fever  curve  with 
extensive  vulvar  changes,  and  death  on  the  13th  day  after  injection. 

This  pig  was  also  bled  on  the  fourth  day  of  fever  and  the  blood 
passed  into  two  normal  animals.  Of  these  guinea-pigs,  one  died  of 
the  disease  and  the  other  survived  and  proved  immune  to  i  c.c. 
of  the  virus  carried  in  the  "Bradley"  strain.  It  was  not  possible 
to  determine  how  many  of  these  ticks  transmitted  the  infection. 

Experiment  V. — This  group  of  ticks  was  also  gathered  in  the 
Lo  Lo  Valley  from  cows.  These  ticks  immediately  attacked  nor- 
mal guinea-pig  2,162;  30  males  and  females  were  found  attached 
to  the  abdomen  inside  of  72  hours.  On  the  fourth  and  succeeding 
days  the  temperature  range  was  104.8°,  105.6°,  105.4°,  97°,  death 
resulting  on  the  eighth  day.  No  external  lesions  were  observed. 
The  spleen  and  inguinal  glands  were  greatly  enlarged.  Blood  was 
transferred  to  normal  guinea-pigs  2,181  and  2,185  ^^^  both  died  of 
spotted  fever.  Blood  from  guinea-pig  2,185  was  injected  into  two 
normal  pigs.  One  of  these  pigs  died  of  spotted  fever,  the  other 
survived  a  typical  and  severe  attack  of  the  disease.  A  subsequent 
immunity  test  with  "Bradley"  passage  blood  proved  that  this  pig 
had  become  immune.  A  "passage  strain,"  using  two  pigs  for  each 
passage,  was  started  with  virus  drawn  from  this  guinea-pig  at  the 
height  of  the  disease.  Fifteen  pigs  in  the  first  eight  passages  died 
with  lesions  typical  of  spotted  fever.  A  survivor  of  the  ninth 
passage  proved  immune  to  a  known  active  virus.  During  the 
following  months  49  passages  of  this  strain  were  made.  Of  the  98 
guinea-pigs  infected  in  this  manner,  in  49  the  disease  was  allowed 
to  run  its  course  without  interference;  of  this  latter  group,  8  pigs 
recovered  from  the  disease,  and  were  thereafter  immune  to  the 
active  virus  which  had  been  obtained  originally  from  a  patient  sick 
with  spotted  fever.  In  more  than  one-half  of  all  these  pigs  hemor- 
rhagic lesions  were  noted. 

To  render  this  series  of  experiments  somewhat  more  complete, 
normal  Montana  ticks  190,  191,  192,  193,  and  195  were  allowed  to 
feed  on  guinea-pigs  infected   with   this   so-called    "natural    tick 


Transmission  of  Spotted  Fever  by  the  Tick  in  Nature    439 

strain."  Ticks  191  and  192  became  infected  and  transferred  the 
disease  to  normal  guinea-pigs  2,394  and  2,398,  thus  completing  a 
tick  cycle  of  this  strain. 

Five  experiments  were  attempted  with  254  ticks  gathered  in 
the  vicinity  of  Pocatello,  Idaho.  Groups  of  50  ticks  were  associ- 
ated with  normal  guinea-pigs.  In  each  case  a  short  period  of  low 
temperature  was  noted.  In  Experiments  III  and  IV  passage  was 
made  to  normal  guinea-pigs  on  the  third  day  of  the  fever.  No 
disease  resulted  and  these  pigs  were  not  immune  to  the  "Bradley" 
strain.  In  the  remaining  three  experiments  of  the  group  no 
conclusive  evidence  of  a  specific  infection  by  the  Idaho  ticks  was 
obtained. 


TRANSMISSION  OF  SPOTTED  FEVER  BY  OTHER  THAN 

MONTANA  AND  IDAHO  TICKS.'-^ 

Maria  B.  Maver. 

(From  the  Pathological  Laboratory  of  the  University  of  Chicago.) 

Experiments  having  shown  that  the  mild  Idaho  type  of  spotted 
fever  may  be  transmitted  by  D.  venustus  of  Montana  and  that  the 
more  virulent  northern  type  of  fever  may  be  carried  by  D.  modestus 
of  Idaho,  it  became  a  matter  of  interest  to  determine  whether  ticks 
of  other  species,  indigenous  in  widely  separated  areas,  might  also 
be  capable  of  acting  as  agents  of  transfer  of  the  spotted  fever  virus. 
In  order  to  carry  out  experiments  on  this  point  arrangements  were 
made  with  several  entomologists  for  supplies  of  wood  ticks  from 
their  localities.  Ticks  were  received  from  Maine,  California,  Texas, 
Utah,  and  Missouri.  The  study  of  these  ticks  as  intermediate 
hosts  for  the  parasite  of  spotted  fever  was  taken  up  according  to 
the  same  plan  as  that  used  in  studying  the  transmission  of  the  virus 
by  the  Idaho  and  Montana  ticks. 

Two  strains  of  the  disease  were  maintained  in  guinea-pigs  at 
the  Chicago  laboratory  at  this  time — the  "Bradley"  strain,  which 
had  arrived  at  the  179th  consecutive  passage  from  the  human 
virus,  and  a  so-called  "natural  tick  strain,"  also  in  guinea-pigs  in 
its  8th  passage,  produced  by  the  bite  of  D.  venustus  (Montana) 
from  nature.     Both  strains  were  used  in  these  experiments. 

I .  Transmission  by  Dermacentor  variabilis  (Say) . — Specimens  of 
this  tick  have  been  found  from  Labrador  to  Florida.  West  of 
the  Mississippi  it  is  not  common.  It  has  been  found  on  a  great  va- 
riety of  small  animals,  more  frequently  on  cattle  and  dogs,  and, 
occasionally,  on  man. 

During  the  month  of  July,  1909,  two  collections  of  tliis  species 
were  received  from  Dr.  Ricketts,  having  been  collected  by  him  in 
the  vicinity  of  Woods  Hole,  Mass.  Fifty  adult  ticks,  the  females 
in  various  stages  of  engorgement,  comprised  these  groups. 

'From  Jour.  Infect.  Dis.,  igil,  8,  p.  327. 

'  This  work  was  supported  by  a  grant  from  the  Memorial  Institute  for  Infectious  Diseases, 
Chicago. 

440 


Transmission  of  Spotted  Fever  441 

Guinea-pig  2,266,  4th  passage  "natural  tick  strain,"  after  an 
incubation  period  of  four  days,  was  placed  in  the  stock,  with  a 
temperature  of  104°  F.  On  the  fifth  day  after  inoculation,  one 
male — No.  i — and  one  female — No.  5 — tick  were  attached  to  this 
guinea-pig  for  48  hours  during  the  period  of  fever.  This  animal 
died  on  the  eighth  day  of  the  disease,  the  autopsy  showing  specific 
spotted  fever  lesions  with  the  hemorrhagic  vulva.  The  impregnated 
female  tick  became  much  enlarged  and  dropped  off  semi-mature 
after  two  days'  feeding.  Any  further  test  of  this  tick  was  impossible 
owing  to  the  fact  that  semi-mature  females  rarely  become  reattached. 
The  female  was  placed  in  a  pill  box  for  oviposition  and  a  later  test 
of  her  larvae  for  inherited  infection  was  made. 

The  male  tick  which  had  fed  on  guinea-pig  2,266  was  placed 
on  normal  guinea-pig  2,284  and  remained  attached  for  72  hours; 
the  temperature  of  this  guinea-pig  remained  normal  during  a 
period  of  18  days  following  attachment.  The  tick  was  found  off 
in  the  sack,  reattached,  and  removed  four  days  later,  in  all  a  period 
of  seven  days'  attachment.  An  immunity  test  given  this  pig  with 
I  c.c.  of  "Bradley"  virus  developed  a  characteristic  case  of  the 
disease.     This  male  tick  did  not  transmit  the  disease. 

Guinea-pig  1,840  of  the  197th  "Bradley"  strain  passage  was 
taken  on  the  third  day  of  fever  with  a  temperature  of  105.6°  F. 
Two  female  ticks — Nos.  8  and  9 — attached  themselves  behind  the 
ears  of  the  pig  after  a  short  period.  This  guinea-pig  died  four  days 
later  with  the  engorged  ticks  still  attached. 

The  female  tick  8  was  immediately  transferred  to  normal  guinea- 
pig  2,321,  and  remained  in  the  same  location  on  this  pig  for  six 
days.  No  disease  developed  in  this  guinea-pig,  and  on  being 
given  I  c.c.  of  "Bradley"  virus  it  died  of  spotted  fever  in  eight  days. 
For  further  maturation  and  impregnation  of  female  No.  8,  male 
No.  I  of  this  group  was  placed  with  her.  Oviposition  began  eight 
days  later.  Examinations  of  the  stained  specimens  of  these  fresh 
eggs  were  made  for  bacilli,  but  no  organisms  were  found.  Exami- 
nations of  eggs  from  normally  developed  ticks  were  also  negative. 
Fifty  fresh  eggs  from  female  No.  8  were  crushed  in  physiological 
salt  solution,  and  injected  into  a  guinea-pig  intraperitoneally,  but 
with  no  production  of  symptoms.    About  600  larvae  were  hatched 


442  Contributions  to  Medical  Science 

from  the  eggs.  These  larvae  matured  on  a  normal  guinea-pig 
without  infecting  it,  26  enlarged  larvae  being  collected  from  this 
pig.  After  their  normal  period  of  moulting,  the  nymphs  were  again 
tested  on  a  normal  pig.  In  no  period  of  their  development  did 
they  transfer  the  disease.  Seven  of  the  eleven  females  matured 
and  deposited  eggs,  hatching  large  numbers  of  larvae. 

From  female  5  about  300  larvae  were  placed  with  infected 
guinea-pig  2,375  of  the  189th  "Bradley"  passage;  35  larvae  fed  on 
this  pig.  Five  infected  guinea-pigs  were  placed  in  this  sack  and 
died  of  the  disease  during  the  feeding  of  these  larvae.  Fourteen 
nymphs  and  19  enlarged  larvae  were  collected.  Normal  guinea- 
pig  2,536  was  infected  by  this  batch  of  nymphs.  The  temperature 
of  the  pig  ran  103°,  105.6°,  105.6°,  104.5°,  and  it  died  on  the  fifth 
day.  On  the  third  day  3  c.c.  of  its  blood  was  withdrawn  and  inoc- 
ulated into  a  normal  and  an  immune  guinea-pig  of  the  natural  tick 
strain.  The  immune  pig  did  not  respond  to  the  virus.  The  normal 
died  on  the  sixth  day  of  fever  with  specific  spotted  fever  lesions. 
Again  blood  was  transferred  from  this  guinea-pig  to  immune  and 
normal  pigs  with  the  same  result;  the  normal  guinea-pig  died  on 
the  eighth  day,  the  immune  showed  no  indication  of  disease. 

The  larvae  from  five  females  were  associated  with  infected 
guinea-pigs  2,371,  2,436,  and  2,482  and  2,311  of  the  "Bradley" 
strain.  These  pigs  died  of  the  disease,  one  after  the  other,  while 
the  larvae  were  feeding.  A  second  test  of  these  nymphs  was  made 
on  normal  guinea-pig  2,563.  Two  of  the  above  larvae  fed  on  this 
pig.  On  the  seventh  day  after  attachment  a  fever  temperature 
began.  The  pig  died  on  the  eighth  day.  Two  passages  of  blood 
were  made  to  normal  pigs  and  to  immune  pigs.  The  normal  pigs 
developed  spotted  fever;   the  immune  pigs  were  unaffected. 

Two  adults  ticks  from  this  group  produced  infection  in  normal 
guinea-pig  2,873.  This  pig  died  on  the  eighth  day  after  tick  at- 
tachment; transfer  of  blood  to  normal  pig  2,915  produced  a  less 
severe  course  of  fever,  which  conferred  immunity  to  the  "Bradley" 
virus. 

2.  Dermacentor  marginatus  Utah  (Banks). — Note  received  with 
these  specimens:  Dallas,  Sub.  ace.  No.  654,  seed  ticks  hatched  from 
eggs  deposited  August  14,  and  days  following.     Hatching  began 


Transmission  of  Spotted  Fever  443 

September  3,  adult  females  taken  from  jack  rabbit,  August  9,  1909, 
at  Milford,  Utah,  by  W.  V.  King. 

The  larvae  were  placed  with  normal  and  infected  guinea-pigs 
in  equal  numbers.  Eight  nymphs  matured  on  the  normal  animal. 
As  a  routine  this  pig  was  given  an  immunity  test  with  "Bradley" 
blood.  This  resulted  in  the  usual  course  of  fever.  Five  infected 
guinea-pigs  were  used  to  mature  and  infect  the  other  half  of  these 
larvae.  After  a  period  of  moulting  four  of  these  nymphs  were 
allowed  to  feed  on  normal  guinea-pig  2,567.  On  the  seventh  day 
after  attachment  a  fever  began,  resulting  in  death  on  the  fifth  day. 
Passage  of  the  heart's  blood  of  this  pig  into  an  immune  and  a  normal 
pig  gave  no  results  in  the  immune  animal  but  produced  typical 
spotted  fever  in  the  normal.  A  second  passage  was  made  with  the 
same  result. 

3.  Amblyomma  Americanum  Linnaeus. — This  species  is  com- 
monly known  as  the  "lone  star  tick"  because  of  the  single  yellow 
spot  on  the  shield  of  the  female.  These  specimens  were  received 
from  Missouri  in  all  stages  of  adult  engorgement.  The  many 
attempts  made  to  attach  these  ticks  to  guinea-pigs  were  unsuccess- 
ful. Nine  of  the  number  were  selected  for  oviposition,  which  was 
evidently  premature  in  every  case.  The  ticks  were  small  and  com- 
paratively few  eggs  were  deposited.  Hatching  took  place  from  the 
eggs  of  females  i,  2,  8,  and  9,  with  about  1,200  larvae  in  all.  Two 
groups  were  made  of  these  larvae,  about  600  in  each,  and  placed 
with  normal  and  infected  guinea-pigs. 

From  two  pigs,  2,410,  2,426,  infected  with  the  "natural  tick 
strain"  100  larvae  fed  and  were  collected  later  from  the  debris  in 
the  sack.  These  enlarged  larvae  developed  in  two  weeks  into 
nymphs,  passing  through  a  moulting  stage.  These  nymphs  were 
associated  with  normal  guinea-pig  2,544  for  a  period  of  two 
weeks  and  eleven  attachments  noted  before  fever  temperature  with 
the  characteristic  hemorrhagic  scrotum  developed.  The  pig  was 
bled  from  the  heart  and  died  on  the  fifth  day.  Two  passages  of 
blood  from  this  infected  pig  to  normal  and  immune  pigs  were  made, 
both  resulting  as  before  in  infection  of  the  normals  and  no  disease 
in  the  immunes.  From  the  14  infected  nymphs,  four  females  and 
two  males  were  attached  to  normal  guinea-pig  2,874.     Three  days 


444  Contributions  to  Medical  Science 

later  the  disease  was  indicated  in  this  pig  by  a  temperature  of  104°, 
reaching  106.8°,  and  the  development  of  hemorrhagic  genitaHa. 
Death  resulted  after  a  sHght  decline  in  temperature  on  13th  day. 
The  ticks  were  removed  after  the  death  of  the  pig  and  immediately 
transferred  to  another  pig.  This  pig  died  on  the  tenth  day,  after 
a  severe  run  of  fever,  with  necrosis  of  the  ears  and  scrotum.  The 
autopsy  in  each  case  showed  typical  spotted  fever  lesions. 

4  and  5. — A  large  number  of  the  larvae  of  the  species  D. 
albipictus  were  dead  when  they  reached  the  laboratory.  About 
50  of  the  Kving  ones  were  placed  with  infected  guinea-pig  2,391. 
Only  one  of  these  was  found  mature  and  died  before  further  test 
could  be  made.  The  larvae  of  the  D.  variabilis  (Utah)  were  in 
poor  condition  when  received.  An  effort  made  to  keep  them  alive 
on  a  normal  pig  failed. 

6.  Ornithodoros  megnini  Duges  (CaHfornia)  was  a  very  interest- 
ing specimen.  Much  time  was  given  to  the  work  of  attaching 
these  ticks  to  rabbits,  without  success.  After  a  latent  period  of 
five  months,  oviposition  took  place,  and  a  great  number  of  larvae 
hatched  out.  When  placed  with  an  infected  guinea-pig  they  im- 
mediately filled  the  ears  and  hung  in  bunches  about  the  face  of  the 
pig.  The  pig  died  in  this  condition.  The  ears  were  cut  off  in  the 
hope  that  these  larvae  would  detach  themselves  later.  This  did 
not  prove  the  case,  and  the  whole  mass  died  attached. 

SUMMARY. 

Ticks  from  six  different  localities  were  received,  and  of  these 
three  groups  developed  so  that  complete  tests  could  be  made,  and 
by  means  of  each  of  these  three,  spotted  fever  was  transmitted 
from  infected  to  normal  guinea-pigs.  In  the  case  of  Dermacentor 
marginatus  (Utah)  and  Amblyomma  Americanum  Linnaeus 
(Missouri)  the  transfer  was  made  with  nymphs ;  no  adult  ticks  were 
tested.  In  the  case  of  Dermacentor  variabilis  (Mass.)  transmis- 
sion of  the  virus  was  effected  by  nymphs  and  later  by  adult  ticks. 

From  these  experiments  it  appears  that  in  so  far  as  abiHty  of 
ticks  from  various  regions  to  transmit  the  virus  of  spotted  fever  is 
concerned,  the  disease  might  find  favorable  conditions  for  its 
existence  in  localities  other  than  those  to  which  it  now  is  Hmited. 


A  CONTRIBUTION  TO  THE  PATHOLOGICAL  ANATOMY 

OF  ROCKY  MOUNTAIN  SPOTTED  FEVER.'- ^ 

E.  R.  Le  Count. 

(.From  the  Pathological  Laboratory  of  Rusk  Medical  College,  Chicago.) 

Six  postmortem  examinations  were  made  by  Dr.  Ricketts  on 
the  bodies  of  persons  dying  of  the  disease.  The  cutaneous  hemor- 
rhages were  general  in  all  and  varied  from  petechiae  no  more  than 
pin-head  sized  to  large  confluent  and  grotesquely  shaped,  bluish- 
black  hemorrhages  covering  all  parts  of  the  body  except  the  palms 
and  soles.  In  a  few  of  the  bodies  hemorrhages  were  observed  in 
the  pleurae  or  omentum,  in  one  instance  in  the  substance  of  the 
lungs.  The  superficial  and  visceral  lymph  glands  were  moderately 
enlarged.  An  icterus  was  always  present.  Enlargement  of  the 
spleen  is  quite  characteristic  of  the  disease.  It  was  from  two  to 
three  times  the  normal  size  in  all  the  bodies  examined.  In  other 
respects  it  was  but  little  altered,  fairly  firm,  and  moderately  hy- 
peremic.  A  large  infarct  was  present  in  one  instance.  The  large 
veins  and  right  chamber  of  the  heart  were  distended  with  blood. 
The  liver  and  kidneys  were  enlarged  and  hyperemic  and  in  both 
organs  varying  degrees  of  cloudy  swelling,  and  fatty  degeneration 
were  met  with.  The  changes  in  the  other  organs  were  neither 
marked  nor  constant  enough  to  deserve  mention,  with  the  excep- 
tion of  the  bone  marrow.  In  two  bodies  where  this  was  examined 
the  marrow  of  the  long  bones  was  red  to  bluish  red. 

In  monkeys  and  guinea-pigs  the  changes  are  like  those  in  man 
with  the  exception  of  the  absence  of  generaHzed  cutaneous  hemor- 
rhages; in  white-skinned  guinea-pigs  these  are  sometimes  seen. 
Localized  hemorrhages  with  necrosis  of  the  scrotum,  vulva,  or 

'  From  Jour.  Infect.  Dis.,  igii,  8,  p.  421. 

'  Several  years  before  the  death  of  Dr.  Ricketts  he  and  I  planned  a  joint  work  on  Rocky  Mountain 
spotted  fever  to  which  he  was  to  contribute  accounts  of  its  etiology,  clinical  aspects,  and  gross  morbid 
anatomy,  and  I  a  study  of  the  minute  changes.  The  plan  dealt  with  a  monographic  consideration  of  the 
disease.  Some  of  the  phases  of  this  work  were  to  be  reported  by  me  at  the  last  meeting  of  the  Association 
of  Pathologists  and  Bacteriologists  in  Washington,  May,  1910,  and  for  this  purpose  he  sent  me  notes  of 
the  gross  changes  he  had  observed  in  human  and  animal  bodies.  No  report  was  made,  news  of  his  death 
arriving  during  the  meeting.    His  observations  are  included  in  the  following  account. 

445 


446  Contributions  to  Medical  Science 

prepuce  are  a  more  constant  result  of  the  disease  in  these  animals; 
occasionally  the  ears  become  necrotic.^  Hemorrhages  are  also  met 
with  in  the  lymph  glands  in  guinea-pigs.  In  other  respects  the 
lesions  of  both  guinea-pigs  and  monkeys  are  quite  like  those  in 
man. 

It  will  not  be  necessary  except  in  a  few  instances  to  compare 
the  microscopic  changes  in  human  tissues  with  those  in  animals; 
they  may  be  considered  as  a  whole.  The  animal  tissues  examined 
were  from  six  monkeys  and  32  guinea-pigs;  the  human  tissues  from 
the  six  bodies  mentioned. 

The  changes  are  of  two  sorts,  those  connected  with  the  occlusion 
of  vessels  and  the  more  diffuse  lesions  affecting  entire  groups  of 
organs.  The  diffuse  changes  are  hyperplasia  of  lymphoid  tissues 
and  cloudy  swelling  and  acute  fatty  changes  in  organs  commonly 
the  seat  of  such  lesions  in  acute  infectious  diseases.  The  focal  lesions 
are  more  varied  in  their  nature  since  they  include  not  only  the  pro- 
cesses leading  up  to  the  occlusion  of  vessels,  but  the  results  of  such 
obstructions,  necrosis  in  different  degrees  and  the  hemorrhages  re- 
sponsible for  so  many  of  the  clinical  and  gross  anatomic  features 
of  the  disease  as  well  as  for  the  name  "spotted  fever."  In  sections 
of  the  skin,  liver,  kidney,  spleen,  and  adrenal  both  vascular  occlu- 
sion and  the  necroses  resulting  from  obstruction  were  present.  In 
the  lung  and  heart  the  capillaries  and  small  veins  were  found 
practically  occluded  with  leukocytes,  but  there  were  no  serious 
consequences  of  these  conditions  with  exception  of  minute  hem- 
orrhages beneath  the  endocardium. 

In  the  least  marked  lesions  of  this  sort  the  vessels  contain  simply 
an  excess  of  small  and  large  lymphocytes  and  some  of  the  latter 
cells  have  as  inclusions  red  cells  or  other  leukocytes.  In  the  more 
obvious  occlusions  agglutinated  red  cells,  fibrin,  and  polymor- 
phonuclear leukocytes  are  also  present.  The  agglutinated  red 
cells  often  are  stained  a  deeper  red  with  eosin  than  adjacent  single 
red  cells.  Fibrin  is  not  demonstrable  in  all  the  sections  of  each 
occluded  vessel,  but  is  intermingled  here  and  there  with  the  other 
constituents.     In  the  human  spleen,  for  example,  and  in  the  monkey 

■  In  the  spotted  fever  of  Idaho  gangrene  occurs  rather  frequently  in  the  h»iman  fauces,  tonsils,  and 
palate,  also  in  the  skin  of  the  penis  and  scrotum;  such  changes  have  not  been  met  with  in  Montana. 


Pathological  Anatomy  of  Spotted  Fever  447 

spleen,  small  sinuses  occur  crowded  to  distension  with  polymorpho- 
nuclear leukocytes,  and  these  obstructed  vessels  form  the  centers 
for  minute  necroses  scattered  throughout  the  spleen  so  abundantly 
that  in  each  field  of  lenses  of  an  amplification  of  100  diameters  or 
thereabouts,  at  least  one  such  lesion  is  present.  The  necroses  re- 
ferred to  of  the  ears  and  skin  of  the  scrotum  in  guinea-pigs  are  due 
apparently  to  anaemia  from  the  plugging  of  small  blood  vessels. 
In  serial  sections  of  ears,  the  seat  of  such  necrosis,  and  made  from 
base  to  the  peripheries,  the  entire  gamut  of  early  and  late  changes 
which  terminate  as  necrosis  are  present  as  well  as  concomitant 
results  of  the  altered  circulation — edema,  hemorrhage,  and  depo- 
sitions of  blood  pigment.  In  the  propagation  of  the  disease  the 
blood  serum  of  an  infected  animal  was  sometimes  injected  into  the 
fresh  animal,  but  the  lesions  described  have  nothing  to  do  with  the 
use  of  ahen  or  homologous  serum  in  the  manner  indicated,  for 
they  are  present  in  the  human  tissues  and  organs  as  well  as  in  those 
of  the  animals  infected  experimentally  by  tick  bites.  A  possible 
exception  is  the  presence  of  rather  compact  masses  of  polymor- 
phonuclear leukocytes  in  the  perivascular  sheaths  of  lymphoid 
tissue  in  the  spleens  of  monkeys,  for  in  this  animal  the  disease  was 
brought  about  by  the  inoculation  of  blood  from  an  infected  guinea- 
pig  or  monkey ;  the  disease  was  not  produced  by  tick  bites  in  any  of 
the  monkeys. 

The  excess  of  leukocytes  in  the  vessels,  which  is  such  a  conspic- 
uous feature  in  different  organs,  is  in  marked  contrast  to  the  esti- 
mations of  the  leukocytes  made  during  Hfe;  in  the  sections  they 
seem  too  numerous  to  be  consistent  with  estimates  of  10  to  14 
thousand  per  cubic  millimeter.^  But  we  are  acquainted  with  a 
similar  condition  in  typhoid  fever  and  in  acute  interstitial  nephritis^ 
— that  is,  an  apparent  disparity  between  the  leukocyte  content  of 
the  blood  during  life  and  the  great  number  in  the  vessels  of  certain 
localities  or  organs.  It  would  seem,  therefore,  as  though  the 
action  of  the  toxin  of  this  disease  was  manifest  in  certain  places, 

•John  A.  Anderson  {Hygienic  Laboratory  Bulletin  No.  14,  igo3,  p.  22)  noted  an  increase  in  the 
large  mononuclears  in  the  blood  of  two  patients  as  the  most  interesting  change  from  the  normal. 

'  Mallory,  "The  Histology  of  Typhoid  Fever,"  Jour.  Exp.  Med.,  1898,  3,  p.  661;  Councilman, 
"Acute  Interstitial  Nephritis,"  ibid.,  p.  393;  Le  Count  and  Batty,  "Purpura  Hemorrhagica  with  General- 
ized Infection  with  Bac.  paratyphosus,"  Jour.  Infect.  Dis.,  1907,  4,  p.  175. 


448  Contributions  to  Medical  Science 

and  at  such  points,  even  though  the  vessel  occlusions  were  not 
present,  there  was  likely  to  be  an  excess  of  leukocytes  in  the  blood, 
mainly  of  the  mononuclear  type. 

No  extensive  search  for  the  bacillus  described  by  Dr.  Ricketts* 
(see  page  368)  was  made.  In  the  examination  of  the  focal  lesions 
it  was  difficult  to  dismiss  the  idea  that  the  cause  of  the  disease  was 
in  all  likelihood  within  the  field  of  vision.  This  was  naturally  more 
true  of  the  minuter  lesions.  The  minuteness  of  the  bacillus,  and 
the  shrinkage  which  is  probably  greater  in  tissues  than  in  cover-glass 
preparations,  as  well  as  its  refractory  staining  peculiarities,  are 
some  of  the  obstacles  to  such  a  search. 

It  is  reasonable  to  believe  that  the  focal  lesions  are  much  more 
widely  spread  than  has  been  demonstrated.  The  bone  marrow 
of  a  few  animals  was  examined  without  finding  any  focal  lesions; 
no  human  marrow  was  obtained.  Similar  necessity  for  further 
study  concerns  the  central  nervous  system.  In  the  few  instances 
in  which  sections  were  examined  of  the  cord  or  brain  (some  of 
these  had  to  do  with  human  tissues)  no  focal  lesions  were  seen. 

In  dismissing  this  phase  of  the  subject  it  is  proper  to  liken  the 
focal  necroses  and  the  prehminary  vessel  changes  to  the  alterations 
caused  by  the  so-called  "endothelial  toxins";  furthermore,  to  recall 
that  some  such  toxins,  it  is  believed,  are  liberated  from  the  bodies  of 
bacteria. 

As  for  the  diffuse  changes,  the  cloudy  swelling  and  fatty  changes 
are  not  marked.  In  the  lymph  glands  an  hyperplasia  occurs  and 
in  the  spleen  a  similar  process  is  in  part  responsible  for  its  consid- 
erable enlargement.  Hemorrhages  are  not  met  with  in  microscopic 
preparations  of  the  lymph  glands  as  frequently  as  in  the  gross  ex- 
aminations. In  two  instances,  both  guinea-pigs  killed  on  the  sixth 
and  seven  days  of  the  disease  respectively,  the  microscopic  prepa- 
rations of  the  lymph  glands  look  not  very  unlike  fiver  at  first  glance, 
due  to  the  great  amount  of  cytoplasm  visible  with  low  powers  of 
the  microscope  and  its  affinity  for  the  cell-body  stains.  These 
large  cells  crowd  and  distend  the  sinuses  and  are  markedly  phago- 
cytic. 

One  of  the  interesting  features  of  the  enlargement  of  the  spleen 

'Jour.  Am.  Med.  Assn.,  igog,  52,  p.  379. 


Pathological  Anatomy  of  Spotted  Fever  449 

in  guinea-pigs  is  the  appearance  of  large  numbers  of  multinucleated 
cells  and  cells  with  nuclei  aggregated  into  rings,  cells  corresponding 
in  some  respects  to  the  giant  cells  of  bone  marrow.  So  far  as 
indicated  by  these  megacaryocyte-Uke  cells,  this  assumption  by 
the  spleen  of  the  characteristics  of  bone  marrow  was  met  with 
in  only  a  few  of  the  spleens  of  guinea-pigs  and  then  early  in  the 
disease.  The  guinea-pig  spleen  normally  contains  such  cells  in 
small  numbers,  but  the  number  present  in  the  spleens  of  some  of 
the  animals  is  very  great.  In  the  human  spleens,  too,  large 
multinucleated  cells  possessing  some  points  of  resemblance  to 
megacaryocytes  are  occasionally  found,  most  of  them  with  several 
small  nuclei  in  the  central  part  of  the  cell,  a  few  with  ring-shaped 
nuclei.  Cells  similar  to  either  the  megacaryocytes  or  to  multinu- 
clear  endothelial  cells  were  absent  from  the  spleens  of  monkeys. 
To  what  extent  these  large  cells  in  the  spleen  are  to  be  attributed 
to  a  myeloid  transformation  of  the  spleen  is  difl&cult  to  state.  We 
are  accustomed  to  attributing  similar  metaplasias  to  severe 
anaemias  of  some  standing. 

In  some  instances,  as  for  example  about  the  vessels  of  the  guinea- 
pig's  seminal  vesicles,  in  and  between  tubules  of  the  testes  and 
epididymites  of  monkeys,  some  of  which  had  not  attained  puberty, 
and  in  other  structures,  the  accumulations  of  cells  and  evidence  of 
their  multipHcation  in  situ  suggest  the  formation  of  new  depots  for 
the  production  of  leukocytes  or  other  cells  which  presumably  are 
in  some  way  designed  to  play  some  role  in  the  defensive  processes. 
These  are  usually  in  perivascular  situations  and  so  Hmited  to  the 
regions  of  the  lymph  channels  that  it  seems  unreasonable  to  ascribe 
them  to  the  focal  processes  of  blood  vessel  obstruction  and  their 
sequences. 

The  evidences  of  obstruction  to  the  outflow  of  bile  are  present 
in  the  human  livers,  more  marked  in  some  than  in  others.  I  have 
not  observed  them  in  the  livers  of  other  animals.  These  changes 
do  not  differ  in  any  noteworthy  way  from  those  encountered  in  the 
livers  of  persons  dying  from  typhoid  fever  or  lobar  pneumonia 
when  an  icterus  develops  during  the  course  of  these  diseases  from 
retrogressive  processes  in  the  liver  cells  and  the  resulting  interfer- 
ence with  the  biliary  current. 


450  Contributions  to  Medical  Science 


EXPLANATION  OF  PLATES. 

(Figs.  lo  [Plate  5],  12  [Plate  6],  15  and  16  [Plate  8]  pertain  to  the  more  diffuse 
lesions,  the  remainder  to  localized  or  focal  processes.) 

Plate  i. 

Fig.  I. — Human  Rocky  Mountain  spotted  fever.  JMinute  necrosis  in  the  spleen. 
X400  diam. 

Fig.  2. — Larger  focal  necrosis  in  the  human  spleen.     X  200  diam. 

Pl.\te  2. 

Fig.  3. — Human  Rocky  Mountain  spotted  fever.  In  the  peripheral  capillaries 
of  the  liver  lobules  leukocytes  are  very  numerous  and  at  various  points  (a)  eosin  stain- 
ing rounded  masses  occur.  In  some  degree  they  resemble  those  met  with  in  typhoid 
fever.     X120  diam. 

Fig.  4. — One  of  the  eosin-staining  ball-like  aggregations,  with  cell  inclusions 
referred  to  in  Fig.  3.     Human.     X400  diam. 

Plate  3. 

Fig.  5. — Experimental  Rocky  Mountain  spotted  fever.  Large  subcapsular 
necrosis  in  the  liver  of  a  guinea-pig.     X  200  diam. 

Fig.  6. — Experimental  Rocky  Mountain  spotted  fever.  Small  region  of  necrosis 
in  the  liver  of  a  guinea-pig.     X400  diam. 

Plate  4. 

Fig.  7. — Thrombosed  vein  in  the  skin.     Monkey.     X250  diam. 
Fig.  8. — Aggregations  of  leukocytes  in  the  perivascular  sheaths  in  the  spleen  of  a 
monkey.     Experimental  Rocky  Mountain  spotted  fever.     X  250  diam. 

Plate  5. 

Fig.  9. — Illustrating  the  necrosis  of  the  guinea-pig's  ear.     X30  diam. 
Fig.  10. — Multinucleated  cells  in  the  human  spleen.     X  700  diam. 

Plate  6. 

Fig.  II. — Illustrating  the  amounts  of  tissue  necrotic  in  some  of  the  livers  of 
guinea-pigs.     X 18  diam. 

Fig.  12. — Numerous  megacaryocytes  in  the  guinea-pig's  spleen.     X80  diam. 

Plate  7. 

Fig.  13. — Aggregations  of  polymorphonuclear  leukocytes  in  perivascular  sheaths 
of  lymphoid  tissue.     Monkey.     X400  diam.     Compare  with  Fig.  8,  Plate  4. 

Fig.  14. — Collections  of  fibrin,  agglutinated  red-blood  corpuscles,  and  leukocytes 
in  the  vessels  of  the  renal  pyramid.     Human.     X4S  diam. 

Plate  8. 

Fig.  15. — Subintimal  proliferation  of  cells.  Human  spleen,  (a),  mitotic  nucleus. 
X400  diam. 

Fig.  16. — Perivascular  accumulations  of  cells  in  the  wall  of  the  seminal  vesicle. 
Guinea-pig.     X  200  diam. 


PLATE    1. 


'•••»    -^    %'-:*•.  ••?'**.*    "•::.; 


Fui.  2. 


PLATE   2. 


1»  » 


■  •  •    .• 


"■'o  •. 


Fig.  3. 


# 


5# 


§>         •' 
6 


bar 


•*  ft  *t 


Fig.  4. 


PLATE   3. 


^K^ 


I'Ki.  r,. 


A 


'^^■^v. 


(/    >    \i^^  3  'd^°-     ^- 


*  I.  J-'- 


*•'  «i^ 


f  * 


1  ^1 


''^  ^  ^^'^ 


/^i  ^'     '■■  rfi     M 


^     ■¥ 


»..  _^ 


*«>  4      9      8  (^ 


^S    y# 


## 


U-*         ^     -^ 


,^t.«K 


KJ     (). 


PLATE   4. 


Fig.  7. 


Fig.  8. 


PLATE  5. 


my^ 


IV 


r^'-. 


Fig.  9. 


Fk;.  10. 


PLATE  6. 


-v^.W. 


W 


.  •'.'V^r^' •*/.'>•••''.  •••••'1 '.'"•/* '-^ 


Fig.  11. 


^:^/;5-// 


»**  'i,*«  '^^\!* 


*^    • 


Ik..  12. 


♦.? 


PLATE   7. 


^mm^^s^^ 


^'^ 


Tft:^^  ^^^  ^ 


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^0 


Fig.  13 


iM.i.    14. 


PLATE  8. 


e.> 

.   ^- 

-n-* 
*          \ 

C 

^ 

IS; 


«^ 


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a- 


^.^•^••^•' 


Fig.  15. 


\' 


I'Ki.   1(3. 


s 


THE  TYPHUS  FEVER  OF  MEXICO  (TABARDH^LO). 

PRELIMINARY  OBSERVATIONS.' 

H.     T.     RiCKETTS    AND    RuSSELL    M.    WiLDEE. 

(Prom  the  University  of  Chicago  [Department  of  Pathology]  and  the  Memorial  Institute  for  Infectious 

Diseases,  Chicago.) 

.  It  will  be  recalled  that  NicoUe^  and  his  associates  recently 
have  greatly  advanced  our  knowledge  of  typhus  fever  by  two 
achievements.  First,  it  was  shown  that  the  disease  could  be 
transmitted  to  the  chimpanzee  by  the  injection  of  the  blood  of 
human  patients,  and  in  a  similar  way  from  the  chimpanzee  to  the 
macacus  monkey  (Macacus  sinicus).  Strangely  they  were  not 
able  to  infect  the  macacus  directly  with  virulent  human  blood. 

Their  second  important  result  consists  in  the  transmission  of 
the  disease  from  macacus  to  macacus  by  means  of  the  ordinary 
body  louse  (Pediculus  vestimenti).  From  the  epidemiologic 
conditions  which  prevail  in  Tunis  they  were  able  to  rule  out  the 
flea  and  the  bedbug  as  carrying  agents. 

When  the  monkey  had  once  been  infected  with  the  louse,  they 
attempted  to  transfer  the  disease  from  animal  to  animal  by  means 
of  injection  from  the  first  monkey,  but  the  experiments  resulted 
only  in  what  were  considered  abortive  attacks  of  t)^hus.  Hence 
they  concluded  that  the  virus  underwent  a  loss  of  virulence  in  the 
monkey. 

They  obtained  apparently  typical  eruptions  in  both  the  chim- 
panzee and  the  monkey,  but  not  uniformly  in  the  latter. 

Recently  also,  Anderson  and  Goldberger^  apparently  have  been 
successful  in  transmitting  the  typhus  fever  of  Mexico  to  a  macacus 
and  a  capuchin  by  the  direct  inoculation  of  virulent  human  blood. 

Their  success  in  this  regard  and  NicoUe's  failure  (i.e.,  to  infect 
the  monkey  by  direct  injections  of  human  blood)  would  seem  to 
bring  into  question  the  identity  of  European  typhus  with  that  of 
Mexico.  Prominent  Mexican  physicians,  indeed,  for  some  years 
have  contended  that  the  two  are  not  identical,  the  chief  difference 

'  From  Jour.  Am.  Med.  Assn.,  igio,  S4,  p.  463. 

'  Compt.  rend.  Acad.  Sc,  1909,  149. 

3  Anderson  and  Goldberger,  Pub.  Health  Rep.,  December  24,  1909. 

451 


452  Contributions  to  Medical  Science 

being  in  the  slower  onset  and  defervescence  of  the  Mexican  disease. 
This  is  particularly  true  in  adults.  In  children  the  onset  and 
defervescence  of  the  Mexican  disease  resemble  the  European 
more  closely. 

It  seems  that  the  relationship  of  the  two  cannot  be  decided 
definitely  without  a  more  detailed  experimental  comparison.  They 
may  be  very  closely  related,  though  perhaps  not  absolutely  identi- 
cal, particularly  in  view  of  the  fact  that  both  are  probably  carried 
by  the  body  louse. 

We  may  now  describe  some  of  our  own  experiments  bearing  on 
the  transmission  of  the  disease  to  the  monkey  and  the  non-filtera- 
bility  of  the  virus. 

I.     transmission  to  the  macacus  by  injection. 

We  have  been  able  to  confirm  the  probable  susceptibility  of 
the  monkey  to  inoculations  with  the  blood  of  patients  suffering 
from  the  tophus  fever  of  Mexico,  as  reported  briefly  by  Anderson 
and  Goldberger,  and  to  obtain  additional  information  regarding 
the  degree  of  susceptibility  of  this  animal. 

In  the  two  monkeys  which  apparently  were  infected  by  Ander- 
son and  Goldberger,  multiple  inoculations  were  used.  Their 
macacus  received  a  total  of  20  c.c,  divided  into  three  injections, 
and  the  capuchin  a  total  of  8  c.c,  in  two  injections. 

In  our  inoculations  single  injections  were  given,  the  animal 
being  Macacus  rhesus. 

On  January  ii  a  quantity  of  blood  was  drawn  from  the  median  basilic  vein  of 
Jose  Hernandez,  a  patient  in  the  Hospital  General  (Mexico  City),  on  the  eighth  day 
of  his  sickness.  The  fever  and  condition  of  the  patient  were  typical  for  an  attack 
of  typhus  of  this  duration,  the  attack  being  one  of  moderate  severity.  Although  the 
skin  was  quite  dark,  the  spots,  not  yet  petechial,  could  be  seen  over  the  abdomen, 
chest,  axillary  skin,  arms,  legs,  and  back,  and  the  conjunctivae  were  reddened  charac- 
teristically. The  spleen  showed  little  or  no  enlargement  on  percussion,  and  it  could 
not  be  palpated.  Since  the  blood  was  drawn  the  patient  has  passed  through  a  ty^pical 
"crisis,"  which  occupied  about  three  days,  and  is  now  convalescent. 

Two  cubic  centimeters  of  the  blood  of  the  patient  planted  in  50  c.c.  of  broth  have 
remained  free  from  discoverable  micro-organisms,  which  corresponds  with  the  usual 
results  of  cultivation  experiments  with  typhus  blood. 

Blood  inoculation  experiment. — Inoculations  of  i,  5,  and  10  c.c.  of  defibrinated 
blood  were  made  respectively  into  monkeys  5,  6,  and  7.  No.  5  died  five  days  after 
its  inoculation,  showing  a  consolidation  of  the  lungs,  and  since  it  had  had  no  fever. 


The  Typhus  Fever  of  Mexico 


453 


it  was  discarded  from  the  experiment.  In  addition,  monkey  4  received  8  c.c.  of  the 
serum  from  the  same  blood. 

The  blood  after  defibrination  stood  at  room  temperature  (15  to  20  C.)  and  in 
diffuse  light,  for  from  six  to  seven  hours  before  injection. 

The  5  c.c.  of  blood  which  morLkey  6,  weighing  2,010  gms.,  received  was  diluted  to 
IS  c.c.  with  sterile  physiologic  salt  solution,  and  the  entire  amount  introduced  intra- 
peritoneally.    Its  temperature  on  successive  days  was  as  follows: 


9:00-10:00 

3:00-4:00 

9:00-10:00 

3:00-4:00 

A.M. 

P.M. 

A.M. 

P.M. 

January  11 

100.9 

January  17 

too.  2 

104.9 

January  12  

99  0 

January  18 

97.6 

104.3 

January  13 

99-3 

IOI.4 

January  19 

103.3 

101.8 

January  14 

100. 1 

102.4 

January  20 

97  0 

101. 0 

January  13 

100.4 

103.6 

January  21 

99  I 

93  o* 

January  16 

97-3 

102.7 

•  Death. 

On  January  17,  six  days  after  inoculation,  the  animal  ate  little,  and  sat  "huddled- 
up,"  with  hairs  more  or  less  erect.  This  condition  continued,  and  on  the  19th  there 
was  increased  secretion  from  the  conjunctivae,  and  the  animal  coughed  moderately. 
The  illness  appeared  more  severe  on  the  20th;  there  was  no  resistance  to  manipulation; 
emaciation;  moderate  diarrhea.  On  the  21st,  when  the  temperature  became  sub- 
normal, the  animal  was  still  somewhat  responsive  until  about  the  middle  of  the  after- 
noon, when  its  condition  grew  rapidly  worse,  and  at  9  o'clock  it  was  moribund. 

The  autopsy,  which  was  performed  at  once,  showed  nothing  distinctive,  and  very 
little  that  appeared  abnormal.  The  lymph  glands  generally  were  moderately  enlarged, 
but  were  not  congested  or  hemorrhagic.  Those  of  the  axilla  and  groin  were  the  seat 
of  old  pigmentation.  The  lungs  were  pink,  and  showed  no  inflammation  or  other 
alteration  except  for  a  slight  amount  of  atelectasis  at  the  upper  border  of  the  left  lower 
lobe.  The  pleurae  were  free  from  signs  of  inflammation.  Moderate  swelling  of  both 
the  kidneys  and  liver  were  present,  but  they  were  not  degenerated.  The  spleen  was 
rather  firm  but  not  distinctly  enlarged.  No  evidence  of  infection  was  found  in  the 
peritoneal  cavity.  The  meninges  and  cerebral  cortex  were  free  from  congestion, 
edema,  or  other  signs  of  inflammation. 

A  bouillon  flask  culture  from  the  heart's  blood,  and  agar  slants  from  the  viscera, 
remained  sterile. 

Monkey  7,  a  male  weighing  2,150  gms.,  received  10  c.c.  of  the  same  defibrinated 
blood,  made  up  to  20  c.c.  with  salt  solution.  One-half  the  quantity  was  injected 
intraperitoneally,  the  other  half  subcutaneouslj'.  Its  temperature  on  successive  days 
is  given  below. 


9:00-10:00 

3:00-4:00 

9:00-10:00 

3:00-400 

A.M. 

P.M. 

A.M. 

P.M. 

January  11 

104.0* 

January  19 

102.2 

10S.2 

January  12 

101.8 

102.6 

January  20 

102.7 

IOS.6 

January  13 

100.8 

102.9 

January  21 

102.4 

103.6 

January  14 

100.2 

102.7 

January  22 

103.2 

106.6 

January  15 

100.  s 

104.1 

January  23 

100.6 

104.8 

January  16 

101.4 

104.3 

January  24 

105.1 

10S.4 

January  17 

98.0 

105. 1 

January  25 

103.2 

104.6 

January  18  

100.4 

104.7 

January  26 

103.1 

107.2 

•  Before  injection. 


454 


Contributions  to  Medical  Science 


The  animal  first  began  to  appear  sick  on  the  i6th,  the  second  day  of  fever.  On 
the  17th  it  made  little  resistance  to  manipulation  and  from  this  on  it  appeared  dis- 
tinctly ill  and  ate  little.  It  developed  no  marked  diarrhea,  although  the  stools  became 
rather  soft.  While  the  temperature  became  high  the  animal  at  no  time  lost  its  respon- 
siveness. The  conjunctivae  were  not  noticeably  reddened,  and  a  distinct  eruption 
could  not  be  identified.     After  a  rather  long  and  severe  course,  the  animal  recovered. 

Monkey  4,  weighing  1,800  gms.,  received  8  c.c.  of  serum  of  the  same  blood;  the 
serum  was  obtained  by  defibrination  and  centrifugation.  This  quantity  was  diluted 
to  25  c.c.  with  salt  solution,  one-half  being  injected  intraperitoneally,  the  other  half 
subcutaneously.     The  temperature  on  successive  days  was  as  follows: 


9:00-10:00 

3:00-4:00 

1 

!    9:00-10:00 

3:00-4:00 

A.M. 

P.M. 

A.M. 

P.M. 

January  11 

103. 6» 

January  n  .  . . . 

104.2 

104.2 

January  12 

100. 0 

January  18  .  .  . 

101  5 

104.2 

January  13 

99-4 

I03-3          I 

January  19 ...  . 

•;        103.2 

103.  s 

January  14 

99-7 

102.1 

January  20 ...  . 

.1        101. I 

103.0 

January  15 

100.4 

103.4 

January  21 ... . 

.  1        100.6 

103.7 

January  16 

101.3 

102.9 

January  22 ... . 

.1           t 
1 

I 

•  Before  injection, 
t  Too  low  to  register. 
%  Death  at  6  p.m. 

On  the  17th,  the  first  day  of  distinct  fever,  the  animal,  which  had  hitherto  appeared 
vigorous  and  healthy,  looked  sick,  unkempt,  its  hairs  stood  up,  and  it  "huddled  up" 
even  in  the  sun.  This  condition  continued,  food  was  refused,  and  emaciation 
developed.  On  the  20th  a  moderate  diarrhea  appeared  and  continued  until  death. 
On  the  afternoon  of  the  2 2d  it  became  soporose,  and  died  at  about  6  o'clock.  An 
eruption  which  could  be  referred  positivelj-  to  the  infection,  or  bearing  a  resemblance 
to  that  of  t>'phus  fever,  could  not  be  identified.  An  "eruption"  which  did  appear 
on  the  skin  of  the  lower  chest  and  the  upper  portion  of  the  abdominal  skin  probably 
was  accidental.  On  the  i8th,  the  second  day  of  fever,  the  monkey  had  been  bled 
from  the  heart,  for  cultivation  and  other  experiments.  A  small  amount  of  blood 
escaped  through  the  skin  when  the  needle  was  withdrawn,  and  at  autopsy  it  was 
found  that  some  subcutaneous  hemorrhage  had  occurred  and  that  the  blood  had 
"diffused"  posteriorly  in  the  form  of  a  rather  narrow  band,  following  the  median  line. 
The  "eruption"  was  roughly  median  although  it  extended  about  an  inch  beyond  the 
visible  limit  of  the  subcutaneous  extravasation.  In  character,  it  was  at  first  pink, 
that  of  the  early  spot,  and  seemed  to  disappear  on  pressure.  It  appeared  two  days 
after  the  heart  had  been  punctured  and  on  the  fourth  day  of  fever.  On  the  following 
day  and  subsequently  it  became  darker,  more  or  less  cyanotic  in  color,  and  could  not 
be  effaced  by  pressure.  The  spots  were  rather  ill-defined,  and  appeared  to  consist  of 
collections  of  minute  punctiform  hemorrhages.  A  similar  condition  could  not  be 
identified  on  other  parts  of  the  bodj',  and  in  view  of  the  subcutaneous  hemorrhage 
which  compUcated  the  situation  it  seems  probable  that  the  eruption  had  its  source 
in  the  latter  rather  than  as  a  manifestation  of  tjiihus  fever. 

At  the  autopsy  the  lungs  were  found  of  a  normal  pink  color;  there  were  no  signs 
of  inflammation.  On  the  visceral  pleura,  particularly  of  the  left  lung,  were  a  number 
of  small,  circular,  dark  red  hemorrhages  from  0.5  to  1.5  mm.  in  diameter.  The 
pleural  cavities  were  normal;    the  heart  normal;    no  inflammation  of  the  valves  or 


The  Typhus  Fever  of  Mexico  455 

pericardium.  The  liver  was  apparently  somewhat  enlarged,  pale,  as  if  fatty,  but 
showed  little  or  no  congestion;  the  lobules  were  well  marked;  anterior  border  dis- 
tinctly rounded.  The  spleen  was  about  i .  5  cm.  longer  than  that  of  monkey  6  and 
perhaps  a  few  millimeters  broader;  was  distinctly  enlarged,  bluish-red  in  color,  and  of 
rather  firm  consistence ;  contained  no  hemorrhages. 

The  kidneys  were  perhaps  a  little  enlarged  and  moderately  congested;  cortex 
and  pyramids  of  a  homogeneous  normal  color;  striations  normal;  the  cortex  had  a 
relation  to  the  medulla  of  about  one  to  one. 

The  mucous  membrane  of  the  colon  was  much  reddened,  and  perhaps  even 
hemorrhagic;  the  colon  contained  a  large  amount  of  glairy  mucus  but  no  blood  or 
feces.  The  mucous  membrane  of  the  ileum  appeared  normal,  and  the  ileum  contained 
nothing  but  a  slightly  viscous  yellowish  fluid;  there  was  a  short  intussusception  with 
no  inflammatory  or  obstructive  signs  of  the  parts  involved.  The  duodenum  con- 
tained bile-stained  mucus,  the  mucosa  being  normal.  The  stomach  contained  some 
undigested  banana  and  mucus. 

The  lymphatic  nodes  everywhere  seemed  more  or  less  enlarged  but  were  not 
congested  or  hemorrhagic.  Those  of  the  groin  and  axilla  were  almost  black  from 
some  previous  pigmentation. 

The  meninges  showed  a  good  deal  of  congestion  and  edema,  the  fluid  being  per- 
ectly  clear.  The  cerebral  cortex  appeared  normal.  Other  parts  of  the  central 
nervous  system  were  not  examined. 

Cultures,  as  in  the  case  of  No.  6,  remained  sterile,  including  those  from  the 
meninges. 

As  a  basis  for  interpreting  these  experiments,  we  have  for  con- 
sideration :  the  existence  of  an  incubation  period  which  was  approxi- 
mately the  same  in  all  three  animals,  and  during  which  they 
remained  healthy;  the  occurrence  of  illness  and  fever  followed 
by  the  death  of  two  of  the  animals;  the  sudden  onset  of  fever  and 
illness,  and  the  rapid  defervescence  in  the  animal  which  recovered ; 
the  negative  outcome  of  cultures;  and  the  more  or  less  negative 
findings  at  autopsy,  corresponding  with  the  condition  in  typhus 
in  man. 

The  temperature  curves  of  monkeys  4  and  6  suggest  that  the 
interpretation  of  infection  purely  on  the  basis  of  the  fever  mani- 
fested may  be  attended  by  diflSculties  in  some  instances.  The 
variations  between  the  morning  and  afternoon  temperatures  of 
uninfected  and  apparently  healthy  monkeys  is  often  considerable, 
and  in  our  experience  has  not  been  constant  in  the  same  animal 
from  day  to  day;  marked  variations  have  been  encountered  when 
different  animals,  apparently  healthy,  were  compared.  The 
morning  temperatures  of  healthy  controls  have  habitually  been 
from  0.8  to  1.8  or  2.0  degrees  lower  than  those  of  the  afternoon. 


456  Contributions  to  Medical  Science 

The  former  is  commonly  below  102.0,  and  may  lie  between  99.0 
and  loi  .0  for  several  days  in  succession;  whereas  in  the  afternoon 
(from  3  to  4  o'clock)  it  commonly  is  foimd  at  some  point  between 
102.0  and  103.0.  The  comparatively  cold  nights  and  the  warmer 
days  may  have  some  influence  on  the  more  extreme  variations. 
We  have  attempted  to  eliminate  this  factor  as  much  as  possible  by 
warming  the  room  from  the  hours  of  5  p.m.  to  9  a.m.,  the  tempera- 
ture at  night  being  about  15  C.  During  the  warmer  portion  of 
the  day  the  animals  have  been  placed  where  they  could  avail 
themselves  of  the  sun. 

It  is  noteworthy  that  No.  6  on  only  one  day,  the  19th,  exliibited 
a  morning  temperature  (103.3)  which  was  distinctly  above  the 
normal  for  that  hour,  and  that  on  only  two  days  after  illness  began 
(17th  and  1 8th)  was  it  distinctly  above  the  normal  for  the  after- 
noon, yet  from  the  17th  on  it  was  manifestly  a  very  sick  animal. 
The  temperature  was  subnormal  for  two  days  preceding  death. 

In  No.  4  the  morning  temperature  was  above  the  normal  Kmit 
on  two  days  (17  th  and  19  th)  and  in  the  afternoon  on  five  successive 
days,  the  period  of  subnormal  temperature  preceding  death  being 
much  shorter  than  in  the  case  of  No.  6. 

In  No.  7  the  morning  temperature  was  somewhat  above  the 
expected  normal  (being  from  102.2  to  103.2)  for  four  successive 
days,  and  in  the  afternoon  there  was  rather  marked  elevation 
for  ten  successive  days. 

The  short  course  of  the  fever  and  the  fatal  termination  in  Nos. 
4  and  6  may  perhaps  be  accounted  for  by  a  relatively  low  resistance 
on  their  part. 

In  drawing  the  line  between  the  end  of  the  incubation  period 
and  the  onset  of  disease,  the  general  appearance  and  behavior 
of  the  animal  appear  to  be  of  equal  or  greater  importance  than  the 
temperature  alone;  taken  together  they  are  a  sufiiciently  satis- 
factory index. 

There  is  some  difference  between  the  incubation  period  of  Nos. 
4  and  6,  on  the  one  hand,  and  No.  7,  on  the  other.  In  the  two 
former  it  lay  between  five  and  six  days,  whereas  in  the  latter  it 
was  reduced  to  four  days  or  less.  The  difference  in  dosage  may 
have  accounted  for  this. 


The  Typhus  Fever  of  Mexico  457 

It  is  worthy  of  comment  that  the  smaller  dose  (5  c.c.)  which 
No.  6  received  seemed  more  virulent  than  the  10  c.c.  administered 
to  No.  7.  Differences  in  the  resistance  of  individuals  may,  of 
course,  be  called  on  to  explain  this.  No.  6  received  its  entire 
injection  intraperitoneally,  whereas  in  No.  7  the  amount  was 
divided  equally  between  the  peritoneal  cavity  and  the  subcutaneous 
tissue.  Whether  there  is  a  difference  in  \drulence  depending  on 
the  route  of  injection  has  not  been  determined.  No.  4  is  hardly 
comparable  with  6  and  7  in  this  regard  inasmuch  as  it  received 
serum  rather  than  defibrinated  blood. 

We  may  further  call  attention  to  the  fact,  as  mentioned  at  the 
outset,  that  a  single  injection  of  virulent  blood,  as  well  as  the 
multiple  injection  used  by  Anderson  and  Goldberger,  is  capable 
of  producing  infection,  and  this  with  a  short  incubation  period. 
It  is  unfortunate  that  the  animal  which  received  i  c.c.  of  the  virus 
died  of  other  causes,  since  it  was  hoped  that  this  injection  might 
furnish  some  index  regarding  the  degree  of  susceptibility  of  the 
monkey.  This  is  important  from  the  point  of  view  of  insect  trans- 
mission, which  would  seem  to  demand  an  animal  of  rather  high 
susceptibility.  The  more  exact  susceptibility  of  the  species  will 
be  studied  further. 

It  is  of  some  significance,  too,  that  the  serum  appears  to  be 
infective  as  well  as  the  defibrinated  blood.  As  yet  we  have  no 
data  concerning  the  relative  infectivity  of  the  two,  further  than 
that  8  c.c.  of  serum  appeared  to  be  as  virulent  for  No.  4  as  5  c.c. 
of  blood  was  for  No.  6. 

The  material  injected  was  diluted  with  salt  solution  on  theo- 
retical grounds,  and  because  experience  with  Rocky  Mountain 
spotted  fever  suggested  that  dilution  of  the  virus  might  favor 
infection.  Dilution  is  known  to  render  less  effective  specific 
antibodies  which  may  be  destructive  to  the  virus,  and  there  can 
be  little  doubt  that  blood  drawn  from  typhus  patients  on  the 
eighth  day  of  the  disease  contains  germicidal  antibodies,  particu- 
larly since  the  disease  is  one  which,  clinically,  is  known  to  cause 
the  development  of  distinct  immunity.  Dilution  may  also  favor 
infection  through  the  peritoneal  route  by  affording  better  condi- 
tions for  rapid  absorption  or  dissemination  of  the  micro-organisms. 


458  Contributions  to  Medical  Science 

In  experiments  with  spotted  fever  we  have  noted  occasionally 
that  smaller  doses  of  virus  would  cause  infection  when  larger 
doses  of  the  same  material  would  not,  the  injections  being  intra- 
peritoneal. Small  doses,  as  o.oi  c.c,  are  usually  diluted  with 
2  or  3  c.c.  of  salt  solution,  whereas  larger  doses,  as  5  ex.,  are  diluted 
to  a  much  less  degree  or  not  at  all. 

The  moderate  diarrhea  which  appeared  in  No.  6  was  probably 
of  no  significance,  inasmuch  as  certain  of  the  uninoculated  animals 
were  similarly  affected.  We  may,  however,  call  attention  to  the 
fact  that  some  Mexican  observers  insist  that  there  is  a  certain 
degree  of  intestinal  disturbance  in  typhus  in  man,  manifested 
frequently  by  diarrhea,  and  the  right  ihac  region  is  habitually 
examined  cHnically  for  signs  of  such  disturbance.  We  have 
observed  such  disturbance  in  a  number  of  instances,  but  have  no 
personal  knowledge  of  its  relationship  to  typhus  fever. 

We  are  hardly  in  position  as  yet  to  make  a  thorough  comparison 
of  the  infection  in  the  monkey  with  that  in  man.  It  may  be 
stated,  however,  that  the  incubation  period  as  obtained  by  this 
method  of  infection  was  shorter  than  that  which  is  accepted  for 
man,  the  latter  being  in  the  neighborhood  of  ten  to  twelve  days. 
Manifestly  this  is  subject  to  considerable  variation  in  experiments, 
since  in  the  macacus  infected  by  Anderson  and  Goldberger  it  was 
twelve  days,  and  in  our  No.  7  only  four  days. 

The  duration  of  typhus  in  man  varies,  according  to  Dr.  Genaro, 
Escalona,  from  about  twelve  days  in  children,  in  whom  it  is  com- 
paratively mild,  to  twenty-one  or  twenty-four  days  in  adults. 
In  monkey  7  the  course  lasted  for  approximately  twelve  days. 

The  onset  in  all  three  animals  was  sudden  and  the  defervescence 
in  No.  7  moderately  rapid,  comparable  to  the  conditions  in  human 
patients.  In  man  the  temperature  is  said  to  rise  gradually  for 
from  three  to  four  days,  when  the  fastigium  is  reached,  the  morn- 
ing remissions  being  marked,  as  in  the  case  of  the  experiments 
described.  In  man  also  defervescence  occupies  from  three  to 
four  days,  followed  by  from  two  to  four  days  of  subnormal  tem- 
perature, corresponding  to  the  conditions  in  monkey  7. 

Bacteriologically  the  conditions  are  similar,  in  that  attempts 
at  cultivation  by  ordinary  methods  have  given  negative  results. 


The  Typhus  Fever  of  Mexico  459 

Likewise  the  findings  at  autopsy  are  similar,  nothing  distinctive 
being  found  in  the  animals,  as  in  the  case  in  human  beings.  The 
cerebral  congestion  and  edema  of  No.  4,  the  slight  or  moderate 
enlargement  of  the  spleen,  congestion  of  the  abdominal  viscera, 
doubtful  or  moderate  enlargement  of  the  lymph  glands,  and  the 
absence  of  localized  inflammations  are  conditions  which  are 
commonly  found  in  man.  The  negative  findings  appear  to  be 
of  more  diagnostic  significance,  when  considered  with  the  clinical 
course,  than  positive  anatomic  changes.  Degeneration  of  the  liver 
and  kidneys  is  frequently  found  in  man,  but  it  is  not  a  necessary 
accompaniment  of  fatal  infections. 

In  the  experiments  performed  so  far,  there  is  a  lack  of  corre- 
spondence with  the  human  infection,  in  that  no  eruption  resembling 
that  of  typhus  appeared  in  the  animals.  Although  it  would  be 
gratifying  to  reproduce  this  phenomenon,  failure  to  do  so  can 
hardly  constitute  a  valid  argument  against  the  character  of  the 
infection  in  the  monkeys.  We  recall  that  not  all  monkeys  and 
guinea-pigs  infected  with  spotted  fever  exhibit  the  eruption,  and 
the  conditions  may  be  similar  in  typhus. 

Eventually  it  seems  probable  that  the  nature  of  the  infection 
in  all  cases  of  recovery  can  be  checked  up  by  means  of  immunity 
tests.  The  disease  appears  to  be  one  which,  in  man,  confers 
distinct  immunity,  and  animals  which  have  once  suffered  from 
infection  should  be  immune  to  further  inoculations.  This  will 
be  investigated  and  reported  on  at  a  future  date. 

II.      FILTRATION   EXPERIMENT. 

With  the  desire  of  obtaining  information  about  the  virus  of 
the  disease,  especially  as  having  a  possible  bearing  on  the  approxi- 
mate size  of  the  micro-organism,  we  have  performed  a  filtration 
experiment  which  it  is  desirable  to  report. 

Of  the  blood  referred  to  in  the  previous  experiments,  34  c.c, 
were  centrifugated  until  the  corpuscles  occupied  approximately 
the  lower  three-fifths  of  the  column.  The  overlying  serum  was 
drawn  off  and  replaced  by  an  equal  amount  of  sterile  salt  solution. 
The  corpuscles  were  thoroughly  mixed  or  washed  with  the  latter 
and  again  centrifugated  moderately,  after  which  the  overlying 


460 


Contributions  to  Medical  Science 


fluid  was  added  to  the  first  portion.  This  was  repeated  again  and 
the  three  fluid  portions  then  combined.  It  seemed  probable  that 
through  this  procedure  one  might  obtain  a  larger  quantity  of  micro- 
organisms in  the  fluid  than  by  resorting  to  a  single  more  vigorous 
centrifugation.  This  seemed  better  also  than  to  attempt  to  filter 
the  uncentrifugated  blood,  which  indeed  is  an  almost  impossible 
task  with  moderate  pressures. 

It  seems  sufficiently  accurate  to  consider  that  the  defibrinated 
blood  consisted  of  about  equal  parts  of  serum  and  corpuscles, 
and  that  we  obtained  the  equivalent  of  about  17  c.c.  of  serum  by 
this  washing  process. 

For  the  purpose  of  filtration  the  serum  was  diluted  to  51  c.c. 
by  means  of  salt  solution,  and  this  quantity  was  divided  into 
two  equal  portions,  one  to  be  filtered  and  the  other  to  be  injected 
without  filtration. 

The  first  portion  was  passed  through  a  small  Berkefeld  candle, 
and  with  the  threefold  dilution  it  filtered  readily.  The  filter  was 
washed  by  passing  through  it  an  additional  5  c.c.  of  salt  solution, 
this  filtrate  being  added  to  the  first. 

The  interval  between  the  drawing  of  the  blood  from  the  patient 
and  the  injections  was  from  six  to  seven  hours,  as  in  the  preceding 
experiments. 

The  total  quantity  of  each  portion  was  approximately  25  c.c, 
of  which  one-half  was  injected  intraperitoneally,  the  other  half 
subcutaneously,  after  suitable  preparation  of  the  skin. 

The  result  of  the  injection  of  the  unfiltered  serum  into  monkey 
4  was  given  in  the  first  part  of  this  paper,  it  being  our  conclusion 
that  the  animal  became  infected  with  and  died  of  tj^phus  fever. 

No.  3,  which  received  the  filtered  serum,  exhibited  the  following 
temperatures  on  successive  days: 


A.M. 

P.M. 

A.M. 

P.M. 

January  1 1 

103.2 

January  18 

102.6 

102.  s 

January  12 

102.  s 

102.6 

January  19 

103.6 

101.6 

January  13 

102.8 

103.3 

January  20 

102.6 

103.6 

January  14 

102.7 

103.6 

January  21 

101.9 

103.9 

January  15 

103.6 

103.8 

January  22 

102.6 

104.4 

January  16 

103 

102.6 

January  23 

101 .6 

102.7 

January  17 

103 

102.8 

January  24 

loi  .7 

103.6 

The  Typhus  Fever  of  Mexico  461 

In  spite  of  rather  high  morning  temperatures,  the  animal  has 
been  in  apparently  perfect  health  since  the  date  of  inoculation, 
which  is  in  distinct  contrast  with  the  course  shown  by  No.  4. 
As  stated  previously,  the  latter,  after  an  incubation  period  of  about 
six  days,  developed  fever,  grew  sick,  and  died  eleven  days  after 
inoculation,  with  findings  which  are  in  harmony  with  those  of 
typhus  fever. 

We  may  therefore  conclude  that  the  virus  did  not  pass  through 
the  filter  employed,  or  if  it  did,  that  it  did  not  pass  through  in 
a  quantity  sufficient  to  produce  recognizable  infection.  It  seems 
probable,  therefore,  that  the  virus  of  the  typhus  fever  of  Mexico 
may  be  classed  with  the  unfilterable.  It  is,  of  course,  to  be  under- 
stood that  the  micro-organisms  may  find  their  way  through  filters 
of  greater  porosity  which  may  be  encountered,  or  through  filters 
with  thinner  walls.  It  is  possible,  too,  that  they  may  even  pass 
through  filters  of  the  type  and  thickness  employed  in  this  experi- 
ment, in  quantities  which  are  sufficient  to  vaccinate,  but  not 
sufficient  to  produce  virulent  infection.  As  affording  an  analogy 
for  this  possibility,  it  may  be  mentioned  that  we  have  on  several 
occasions  produced  extremely  mild  attacks  of  spotted  fever  in  the 
guinea-pig,  resulting  in  permanent  immunity,  by  the  injection  of 
very  small  doses  of  virus.     This  possibihty  will  be  investigated. 

m.     THE     QUESTION    OF    CONTAGIOUSNESS    AND     TRANSMISSION    BY 

INSECTS. 

Although  the  old  idea  that  typhus  is  a  contagious  disease  still 
has  strong  supporters  in  Mexico  City,  the  conception  that  insect 
transmission  is  in  better  accord  with  the  epidemiologic  conditions 
is  prominent  in  the  minds  of  those  who  are  in  daily  contact  with 
the  disease. 

Without  giving  a  detailed  presentation  of  the  conditions  at 
this  time,  it  may  be  stated  that  of  the  three  insects  which  are  most 
open  to  suspicion,  i.e.,  the  louse  (Pediculus  vestimenti),  the  bedbug, 
and  the  flea,  only  the  first  would  seem  to  merit  serious  considera- 
tion, because  of  the  epidemiology  of  the  disease.  This  conclu- 
sion is,  of  course,  strongly  supported  by  the  results  already  obtained 
by  Nicolle. 


462  Contributions  to  Medical  Science 

The  season  of  greatest  predominance  of  typhus  in  Mexico 
City  (the  spring)  does  not  coincide  with  the  period  of  greatest 
prevalence  of  the  flea  (summer).  And,  regarding  the  bedbug,  we 
have  knowledge  of  individual  cases  which,  it  would  seem,  could 
not  possibly  have  been  carried  by  this  insect. 

Although  our  own  experiments  with  the  louse,  which  have  been 
under  way  for  some  time,  have  not  yet  resulted  in  transmission 
they  will  be  carried  further  and  reported  in  detail  at  a  future  date. 

We  take  pleasure  in  acknowledging  our  great  indebtedness  to  the  local  authori- 
ties and  physicians  of  Mexico  City  who  have  co-operated  with  us  in  various  ways: 
more  particularly  to  Dr.  Gavino,  Director  of  the  Bacteriologic  Institute,  where  we 
enjoyed  the  advantage  of  a  well-equipped  laboratory,  and  to  his  assistant,  Dr.  Girard; 
as  well  as  to  Drs.  Fernando  Lopez  and  Genaro  Escalona,  of  the  General  Hospital. 


1 


THE    TRANSMISSION    OF    THE    TYPHUS    FEVER    OF 

MEXICO    (TABARDILLO)    BY    MEANS    OF    THE 

LOUSE  (PEDICULUS  VESTIMENTI).^ 

H.    T.     RiCKETTS    AND    R.     M.    WILDER. 

{From  the  Department  of  Pathology  of  the  University  of  Chicago  and  the  Memorial  Institute  for  Infectious 

Diseases,  Chicago.) 

In  a  previous  article^  we  referred  to  Nicolle's  experiment,  in 
which  he  apparently  succeeded  in  transmitting  the  typhus  fever 
of  Tunis  from  the  chimpanzee  to  the  macacus  monkey,  through 
the  bite  of  the  louse.  Nicolle  used  twenty-nine  lice  in  his  experi- 
ment; after  feeding  on  the  chimpanzee  they  were  allowed  to  feed 
on  the  monkey  for  several  successive  days.  Also,  Anderson  and 
Goldberger^  report  two  attempts  to  transmit  the  typhus  fever  of 
Mexico  City  to  the  macacus  by  means  of  the  louse.  Their  first 
experiment  seemed  to  be  without  result,  and  although  the  second 
showed  a  slight  rise  in  temperature,  their  inability  to  give  an 
immunity  test  to  the  animal  makes  it  impossible  to  interpret  the 
result.  This  test  is  essential,  as  will  be  manifest  from  consid- 
erations which  follow. 

Since  January  i  we  have  performed  a  series  of  experiments 
bearing  on  the  relation  of  Pediculus  vestimenti  to  the  typhus  of 
Mexico,  some  of  which  may  be  recorded  at  this  time. 

Certain  conditions  pertaining  to  the  experiments  should  be 
detailed  before  the  protocols  are  given. 

The  first  relates  to  the  susceptibiHty  of  the  monkey  to  typhus, 
and  the  positiveness  with  which  the  disease  may  be  recognized 
in  this  animal. 

As  shown  by  the  experiments  of  Anderson  and  Goldberger, 
and  by  our  own,  the  injection  of  virulent  blood  from  man,  under 
suitable  conditions,  gives  rise  to  a  well-marked  course  of  fever, 
following  an  incubation  period  from  five  to  twelve  days.  A  further 
constant  factor  is  the  absence  of  micro-organisms,  which  are  sus- 

■  From  Jour.  Am.  Med.  Assn.,  igio,  54,  p.  1304. 

'Ibid.,  p.  463.     See  p.  451  of  this  book. 

'  Anderson  and  Goldberger,  Pub.  Health  Rep.,  February  18,  1910. 

463 


464  Contributions  to  Medical  Science 

ceptible  to  cultivation  by  ordinal  methods,  from  the  blood  of 
the  animal  during  his  course  of  fever.  This  sequence  of  events 
has  now  resulted  a  sufficient  number  of  times  without  any  failures, 
so  that  it  may  be  expected  constantly  when  the  established  technic 
is  observed.  In  the  course  of  our  experiments  so  far,  six  monkeys 
have  received  virulent  blood  from  man  in  doses  of  from  i  to  7  c.c; 
two  have  received  virulent  unfiltered  serum  in  doses  of  7 . 5  and  8 . 5 
c.c;  and  one  received  5  c.c.  of  defibrinated  blood  from  another 
monkey  as  an  experiment  in  passage.  All  of  these  animals  exhib- 
ited an  incubation  period  followed  by  a  distinct  course  of  fever, 
accompanied  by  signs  of  severe  illness,  and  with  negative  attempts 
to  obtain  cultures  from  the  blood.  Anderson  and  Goldberger 
obtained  similar  results  in  the  four  animals  inoculated  by  them. 
It  would  seem,  therefore,  that  even  in  the  absence  of  an  eruption 
resembhng  that  of  typhus  (and  indeed,  no  such  eruption  has 
appeared),  the  constancy  of  the  phenomena  mentioned  is  a 
satisfactory  index  of  the  transmissibility  of  the  disease  to  the 
monkey.  Constantly  the  temperatures  of  seven  or  eight  normal 
monkeys  were  taken  twice  daily  as  a  means  of  detecting  possible 
adventitious  infections  which  might  be  contagious  in  character; 
but  none  was  observed. 

A  second,  and,  as  it  seems,  a  conclusive  method  of  determining 
whether  a  course  of  fever  was  really  due  to  an  infection  with 
typhus,  consists  of  an  immunity  test,  i.e.,  a  second  injection  of 
virulent  blood  after  the  subsidence  of  the  fever.  The  immunity 
test  must,  of  course,  be  accompanied  by  the  injection  of  a  similar 
quantity  of  the  same  blood  into  a  normal  monkey,  as  a  control 
to  the  virulence  of  the  material.  Thus,  six  monkeys  (Nos.  3, 
7,  9,  II,  24,  and  25)  which  exhibited  a  distinct  course  of  fever 
following  their  first  inoculation,  and  all  of  which  were  seriously 
ill,  showed  no  febrile  response,  or  other  signs  of  illness,  when  the 
second  inoculations,  or  the  immunity  tests,  were  given.  They 
received  their  immunity  tests  in  three  groups  at  different  times 
and  in  all  five  normal  controls  were  used  for  each  group.  Five 
c.c.  of  human  virus  were  injected  into  each  member  of  one  of  the 
groups,  and  4  c.c.  into  each  of  the  other  two.  The  controls  in  each 
case  developed  a  course  of  fever  similar  to  that  already  described. 


Transmission  of  Typhus  Fever  of  Mexico  by  the  Louse    465 

The  degree  of  susceptibility  of  the  monkey  is  another  important 
consideration,  and,  if  it  is  low,  experiments  on  insect  transmission 
might  be  rendered  difficult  or  impossible.  It  will  be  recalled  that 
NicoUe  failed  entirely  in  his  attempts  to  infect  the  macacus  with 
the  Old  World  typhus  by  the  direct  injection  of  human  blood;  he 
succeeded  only  by  first  infecting  the  chimpanzee  and  then  trans- 
ferring blood  from  the  latter  to  the  macacus.  After  the  macacus 
was  once  infected,  he  attempted  to  maintain  the  disease  in  this 
species  by  passage,  but  the  virus  apparently  underwent  a  rapid 
loss  in  virulence,  and  the  infection  "died  out"  after  two  or  three 
passages. 

It  seems  also  that  Anderson  and  Goldberger  had  a  similar 
experience  in  trying  to  maintain  Mexican  typhus  in  the  macacus. 

We,  also,  have  made  two  attempts  to  keep  the  disease  alive 
by  passage.  In  one  instance,  the  second  monkey  (No.  11)  became 
infected,  whereas  the  third  (No.  22)  showed  no  fever.  In  the 
second  instance  the  second  animal  (No.  9)  exhibited  only  an  in- 
definite fever,  and  further  passage  was  not  attempted. 

There  was,  therefore,  sufficient  reason  to  suspect  that  the 
macacus  might  be  sufficiently  susceptible  for  satisfactory  work 
on  insect  transmission.  Information  of  at  least  suggestive  value 
regarding  this  question  might  be  obtained  by  determining  the 
minimum  infective  dose  of  human  blood  for  the  monkey.  Although 
it  appeared  inadvisable,  on  account  of  the  cost,  to  investigate  this 
point  extensively,  one  experiment  was  performed  in  which  one 
animal  (No.  25)  received  i  c.c.  of  virulent  human  blood,  and  another 
0.2  c.c.  of  the  same  material.  After  the  lapse  of  an  incubation 
period  of  ten  days,  the  former  developed  a  severe  course  of  fever 
of  ten  days'  duration,  and  showed  no  reaction  to  a  second  injection 
which  was  given  a  month  later.  The  animal  which  received  o .  2 
c.c.  had  no  fever  and  showed  no  signs  of  illness.  Although  the 
immunity  test  of  the  latter  remains  to  be  given,  it  is  probable 
that  the  minimum  infective  dose  in  this  instance  lay  between 
o.  2  c.c.  and  i  c.c. 

The  question  arises,  however,  as  to  whether  the  monkey  may 
undergo  a  mild  type  of  infection,  which  might  be  accompanied 
by  little  or  no  fever,  and  which  would  be  recognizable  only  from 


466 


Contributions  to  Medical  Science 


the  fact  that  the  mild  or  ''abortive"  attack  had  established  immu- 
nity to  a  subsequent  injection  of  virulent  blood. 

The  possibility  of  such  an  occurrence  is  well  known.  We  have 
observed  it  a  number  of  times  in  experiments  with  spotted  fever 
on  the  guinea-pig.  We  have,  furthermore,  one  instance  of  a  very 
mild  and  scarcely  perceptible  infection  with  typhus  in  the  second 
attempt  at  passage  referred  to  above. 

Monkey  4,  which  was  infected  with  virulent  hximan  serum,  and  whose  history 
was  given  in  a  previous  paper,  was  bled  from  the  heart  on  its  second  day  of  fever  and 
5  c.c.  of  the  blood  were  injected  intraperitoneally  into  No.  9,  which  showed  on  succes- 
sive days  the  temperature  given  in  Table  i : 

TABLE  I. 
Temperature  of  Monkey  9  atter  Intraperitoneal  Injection  of  Infected  Blood. 


AM. 

P.M. 

F. 

c. 

F. 

c. 

103.6 
102. 5 
101.8 
103.2 
102.3 
102.0 
101.6 
102.0 
102.0 
loi  .0 
102.7 
102.9 
101.8 
102.9 
102.7 
104.0 
104.0 
J03O 
103. 1 
101.8 

lOI.O 

39.8 
39-2 
38.8 
39  S 
391 
38.9 
38.6 
38.9 
38.9 
38.3 
39-3 
39-4 
38.8 
39-4 
39-3 
40.0 
40.0 
39-9 
39-5 
38.8 

38:3 

102.6 
103.4 
103.1 
103.9 
102.8 
102.  s 
102.6 
103.6 
103.2 
102.4 
104 -3 
102.0 
103.1 
103.4 
103.2 
104.8 
104-4 
103.9 
103.6 
103 -3 
103.0 
102.8 

January  20 

39.6 

January  23 

39-3 
39.  2 

January  26 

January  27 

39-8 
39  S 

40.2 

38.9 

January  31 

February  i 

39S 
39-7 

40.4 

February  4 

40.2 

39-9 

February  6 

3Q.8 

February  7 

39.6 

February  9 

39-3 

Below  103  for  two  weeks  thereafter. 

Not  until  the  sixteenth  day  after  inoculation  did  the  temperature  rise  above  a 
possible  normal  (for  this  individual),  remaining  somewhat  elevated  for  only  three 
or  four  days.  A  positive  diagnosis  of  infection  with  tjqphus  could  not  be  made  under 
these  conditions;  yet,  when  an  immunity  test  was  given  (February  18)  the  tempera- 
ture did  not  rise  higher  than  103  during  the  twenty-six  days  it  was  under  observation. 
The  control  (No.  18)  exhibited,  on  the  other  hand,  a  severe  course  of  fever. 

Hence,  as  a  consequence  of  this  experiment,  and  from  the 
experience  with  spotted  fever,  it  seemed  not  unreasonable  to 
anticipate  that  the  monkey  might  suffer  from  mild  infections 
with  typhus,  not  manifested  by  marked  elevation  of  the  tempera- 
ture or  by  distinct  signs  of  illness,  but  the  occurrence  of  which 


Transmission  of  Typhus  Fever  of  Mexico  by  the  Louse    467 

might  be  determined  later  by  means  of  immunity  tests.  This, 
we  believe,  is  what  happened  in  our  attempts  to  transmit  the 
disease  by  means  of  the  louse,  as  will  appear  below. 

The  point  may  also  be  raised  as  to  whether  some  of  our  monkeys 
may  have  acquired  typhus  by  contagion,  or  by  means  of  their 
own  lice  from  other  monkeys  which  had  been  infected  deliberately, 
thus  vitiating  later  experiments.  Although  the  evidence  is  entirely 
against  t^'phus  being  contagious,  we  protected  our  experiments 
(by  first  killing  the  lice  on  the  monkeys  by  means  of  insect  powder, 
petroleum,  and  scrubbing  with  green  soap;  and  furthermore,  by 
the  practice  of  keeping  a  healthy  monkey  in  the  same  cage  with  an 
infected  animal  in  order  to  test  the  question  of  contagiousness). 
Two  monkeys  which  were  thus  exposed  (Nos.  18  and  20)  proved 
susceptible  to  later  inoculation,  the  result  tending  to  disprove 
contagiousness. 

Comparatively  large  numbers  of  lice  were  used  in  each  experi- 
ment, because  of  the  supposed  low  susceptibility  of  the  monkey. 
This  condition  lays  the  experiments  open  to  the  objection  that  the 
transmission  might  be  purely  mechanical,  and  may  not,  on  this 
account,  reproduce  the  natural  conditions.  The  natural  method, 
however,  is  still  unknown,  and  may  indeed  be  of  a  mechanical 
nature  rather  than  the  so-called  "biologic."  If  sixty  lice  are  able 
to  infect  a  monkey  mechanically,  a  smaller  number  may  infect 
man,  provided  the  latter  is  more  susceptible.     Protocols  follow. 

Transmission  of  Typhus  from  Man  to  the  Monkey  by  Means  of  Pediculus 

vestimenti. 

Group  3,  Pediculus  vestimenti,  was  infected  as  follows:  January  5,  they  fed 
for  thirty  minutes  on  E.  S.,  on  the  tenth  or  eleventh  day  of  his  sickness;  January  6, 
on  J.  v.,  tenth  day  of  sickness;  January  7,  on  C.  A.,  ninth  day  of  sickness;  January 
8,  on  the  same  patient. 

On  January  9,  45  lice  were  alive  and  40  fed  on  monkey  i  for  about  one  hour: 
January  10,  334  were  alive  and  23  fed  on  the  monkey;  January  11  they  were  not 
fed;  January  12,  19  fed;  January  13,  13  fed;  January  14,  11  fed;  January  15,  9  fed. 
The  feedings  were  not  carried  farther. 

The  temperature  of  the  monkey  on  successive  days  was  as  shown  in  Table  2 . 

It  will  be  noted  that  in  the  afternoons  of  January  17,  20,  21,  and  22,  the  tem- 
perature lay  between  103.2  and  103.6,  the  first  rise  being  eight  days  after  the  lice 
began  to  feed,  or,  having  the  20th  in  mind,  the  interval  was  ten  days.  Inasmuch  as 
the  animal's  temperature  was  constantly  below  102 . 5  for  a  period  of  thirty-one  days 


468 


Contributions  to  Medical  Science 


later,  it  is  not  improbable  that  the  slight  elevation  referred  to  represented  a  mild 
infection,  although  no  other  signs  were  apparent. 

TABLE  2. 
Temperature  of  Monkey  i  apter  Being  Bitten  by  Infected  Lice. 


4 


A.M. 

F.U. 

F. 

c. 

F. 

C. 

I00.4 

I02.8 

98.6 

99.2 

99-4 

101.2 

100.8 

99.0 

100.6 

100.6 

90-1 

98.9 

101 .2 

99-7 

100.9 

99-4 

98.5 

38.0 

39-3 
37.0 
37.3 
37.4 
38.4 
38.2 
37.2 

38.1 
37.3 
37-2 
3S.4 
37.6 
38.3 
37.4 
37-5 

104.6 
101.2 
102.9 
99-9 
loi  .7 
100.2 
103 -6 
102.9 
102.0 
103.2 
103.2 
103.2 
102.6 
102. 1 
102.6 
103.3 

40.3 

38.4 

39-4 

37-7 

38.7 

37  -9 

39-8 

39-4 

38.9 

30  S 

395 

395 

39.3 

39.0 

39.2 

39.6 

Did  not  rise  above  102 . 4  F.  thereafter. 

The  belief  that  the  animal  had  suffered  from  typhus  was  corroborated  by  an  im- 
munity test,  consisting  of  7  c.c.  of  defibrinated  blood  from  patient  27,  given  February 
8.  For  nineteen  days  thereafter,  the  morning  temperature  lay  between  99.0  and 
102.0,  and  in  the  afternoon  between  101.5  and  102.8.  This  immvmity  test  was 
controlled  by  inoculations  of  4  c.c.  of  the  same  blood'  into  No.  3,  and  i  c.c.  into 
No.  25. 

No.  3,  after  an  incubation  period  of  seven  days,  and  No.  25  of  nine  days,  developed 
fever,  which  in  both  animals  lasted  for  ten  days,  going  as  high  as  106  in  the  former, 
and  106.8  in  the  latter. 

The  immunity  of  No.  i  was  again  tested  a  month  later,  March  11,  with  the  same 
result,  whereas  control  20,  after  an  incubation  period  of  five  or  six  days,  passed  through 
the  usual  course  of  high  fever.  The  first  immunity  test,  however,  is  the  one  which 
indicates,  and  it  would  seem  conclusively,  that  No.  i  was  infected  by  the  lice. 


Transmission  from  Monkey  to  Monkey  by  Means  of  the  Louse. 

Monkey  7  was  infected  by  the  injection  of  blood  from  man,  as  described  in  our 
first  paper. 

Group  5,  Pediculus  vestimenti,  was  infected  by  three  feedings  on  monkey  7,  on 
the  sixth,  seventh,  and  eighth  days  of  its  fever.  Thereafter,  the  lice  were  fed  for 
eight  successive  days  on  monkey  12,  their  number  gradually  decreasing  from  eighty- 
one  on  the  first  day  to  nine  on  the  last  day  of  feeding. 

The  temperature  of  No.  1 2  was  irregular  and  rather  high  prior  to  the  experiment, 
although  the  animal  was  active  and  well  nourished. 

'  No.  3  had  been  injected  p^e^'iously  with  filtered  serum,  without  becoming  infected;  hence  this 
injection  constitutes  an  immunity  test  for  No.  3. 


Transmission  of  Typhus  Fever  of  Mexico  by  the  Louse    469 


Its  temperature  continued  as  in  Table  3: 

TABLE  3. 
Temperatukje  of  Monkey  12  after  Being  Bitten  by  Infected  Lice. 


F. 


C. 


January  23 
January  24 
January  25 
January  26 
January  27 
January  28 
January  29 
January  30 
January  31 
February  i 
February  2 
February  3 
February  4 
February  s 
February  6 
February  7 
February  8 


103.4 
103.6 
102.7 
102.6 
102.9 
104.2 
103.3 
103.0 
102.7 
103.7 
103.7 
103.6 
103-4 
102.1 


103.2 
103.6 
103.4 
103.9 
104.3 
103.2 
loi  .9 
103.0 
104.2 
104.0 
103.6 
103.4 
103.3 
104.0 
103.  S 
103.7 
103.7 


A  definite  course  of  fever  cannot  be  made  out  positively.     If  present,  it  would 

appear  to  lie  between  January  30  and  February  5  or  6,  a  period  of  seven  or  eight 

days,  and  the  incubation  period  would  be  seven  or  eight  days.     On  February  2,  the 

animal  was  manifestly  ill,  in  contrast  to  its  former  active  condition,  and  this  condition 

continued  for  four  or  five  days.     On  February  9,  however,  it  was  again  active  and 

appeared  well.    At  this  time  an  immunity  test  was  given;  the  temperature  is  given 

in  Table  4: 

TABLE  4. 

Temperature  of  Monkey  12  after  Immxtnity  Test. 


A.M. 

P.M. 

F. 

C. 

F. 

c. 

February  9 

103.9 
102.5 
103.3 
103.1 
102.9 
102.9 
102.7 
102.3 
102.9 
103.2 
103.2 
IOI.9 
102.9 
102.6 
102.4 

lOI.I 

102.9 
102.4 
102.4 

103.9 
103.9 
103.9 
103.7 
103.4 
103.4 
104-3 
104.4 
103.  S 
103.  S 
104. 1 
103.7 
103.6 
102.8 
102.9 
103.2 
102.4 
103.7 
102.6 

39.9 
39.9 
39.9 
39.8 
36.7 
39.7 

40.  a 
40.3 
39-7 
39.7 
40.1 
39-7 
39-7 
39-3 
39-4 
39.5 
39-1 
39-7 
39-2 

39 
39 
39 
39 
39 
39 
39 
39 
39 
39 
38 
39 
39 
39 
38 
39 
39 
39 

2 
6 

5 
4 
4 
3 
I 
4 
S 
S 
8 
4 
2 
Z 
3 
3 
I 

February  12 

February  15 

February  25 

Continued  similarly  for  twelve  days  more. 

The  controls  were  Nos.  3,  24,  and  25,  already  cited.  A  second  immunity  test 
resulted  in  the  same  way,  No.  20  (see  above)  being  the  control.  During  neither  of 
the  immunity  tests  did  the  animal  show  any  sign  of  illness. 


470 


Contributions  to  Medical  Science 


No.  225  is  a  particularly  good  control  for  No.  12,  since,  like  the  latter,  its  tempera- 
ture was  naturally  high  and  irregular.    The  course  of  fever  was  as  shown  in  Table  5 : 

TABLE  s- 
Temperature  of  Monkey  25,  Control  of  No.  12. 


A.M. 

P.M. 

F. 

c. 

F. 

C. 

103.9 
104.0 
103.3 
103 -9 
103.5 
102.  s 
100.9 
103.4 
102.3 
102.  s 
104.6 
103.7 
104.4 
104.2 
106.3 
106.2 
104.6 
105.8 
103.6 
101.6 

40. 
40. 

39-6 

40. 

39-7 

III 
39-7 
39- 1 
39-2 
40.3 
39.8 
40.2 
40.1 
41-3 
41.2 
40-3 
41 .0 
39-8 
38.7 

104.4 
104.0 
104.1 
103.2 
103.6 
103-3 
103.9 
103.9 
103.2 
105.2 
105.5 
104.4 
104.4 
104.7 
106.8 
105.8 

104. 5 
104.2 
103.2 
102.9 

40.2 

40.0 

40.1 

39-6 

39-8 

39  6 

40.0 

40.0 

39-6 

40.7 

40.8 

40.2 

40.2 

40.4 

41.5 

41 .0 

40.3 

40.1 

39. 5 

39-4 

Continuing  as  before  the  fever  appeared. 

In  our  opinion,  the  result  justifies  the  conclusion  that  No.  12  was  infected  by  the 
lice  of  Group  5. 

Infection  of  the  Monkey  by  Introducing  Intestinal  Contents  of  Lice  into 

Scarifications. 

A  first  experiment,  which  consisted  of  the  subcutaneous  injection  of  the  intestinal 
contents  of  infected  lice,  resulted  in  death  in  less  than  twenty-four  hours  from  sep- 
ticemia. 

A  second  experiment  was  performed  as  follows:  As  the  lice  of  Group  5  were 
feeding  on  monkey  12,  a  small  quantity  of  feces  was  collected  from  a  number  of  the 
lice  as  it  was  extruded,  and  placed  in  a  sterile  test-glass.  To  this  material  the  abdomi- 
nal contents  of  three  lice  were  added  and  the  mass  was  triturated  in  sterile  salt  solu- 
tion.    This  was  done  three  days  after  the  last  feeding  of  the  Hce  on  infected  monkey  7. 

Twelve  small  incisions,  each  less  than  one-eighth  inch  in  length,  and  extending 
through  the  entire  depth  of  the  skin,  were  made  in  the  abdominal  skin  on  monkey 
13.  The  emulsion  of  feces  and  abdominal  contents  was  then  instilled  into  these 
incisions,  which  thereafter  were  massaged  by  means  of  a  sterile  probe.  The  incisions 
healed  promptly  and  without  suppuration.  The  temperature  of  No.  13  was  as  shown 
in  Table  6 . 

As  appears  in  the  table,  the  temperature  rose  on  the  fifth  day  after  inoculation 
and  remained  above  the  normal  for  this  animal  for  five  or  six  days.  During  this 
period  the  animal  became  passive  and  was  not  inclined  to  run  about,  although  it 
was  not  seriously  ill  at  any  time. 

As  a  consequence  of  an  immunity  test  given  on  February  9,  the  animal  showed 
no  febrile  reaction  whatever  and  appeared  perfectly  well,  whereas  the  controls  (Nos. 


Transmission  of  Typhus  Fever  of  Mexico  by  the  Louse    471 


3,  24,  and  25)  reacted  with  severe  fever,  as  stated  above.  A  second  immunity  test, 
given  a  month  later,  gave  the  same  results,  the  control  in  this  instance  being  No.  20 
(see  above). 

TABLE  6. 
Temperature  of  Monkey  13  after  Injection  with  Abdominal  Contents  of  Infected  Lice. 


A.U. 

P.U. 

F. 

C. 

F. 

C. 

102.7 
102.4 
100.3 
102.2 
102.8 
103.3 
103.0 
102.2 
104.4 
103.7 
102.4 
101.8 
102.0 

103-4 
103.6 
102. 1 
IOI.9 
104.0 
103.9 
103.8 
103.8 
102.4 
103.0 
103. 1 
102.  I 
103.0 

39.6 
39.7 
390 
38.8 
40.0 
40.0 
39-8 
39-8 
39.1 
39-4 

39. S 

390 
39-4 

39 
38 
39 
39 
39 
39 
39 
40 
39 
39 

3§ 
38 

I 
0 
0 
3 
6 
4 
0 
2 
8 
1 
7 
8 

February  3 

February  s 

February  6 

February  7 

In  our  judgment,  this  experiment  proves  the  existence  of  the  virus  of  typhus 
fever  in  the  abdominal  contents  of  the  louse  for  at  least  three  days  after  feeding  on 
infected  blood. 

SUMMARY  AND   CONCLUSIONS. 

1 .  It  seems  that  Macacus  rhesus  can  be  infected  with  tabardillo 
invariably  by  the  injection  of  virulent  blood  from  man  taken  on 
the  eighth  to  tenth  day  of  fever.  The  blood  should  be  diluted  with 
salt  solution,  as  stated  previously. 

2.  Attempts  to  maintain  typhus  in  the  monkey  by  passage 
through  other  monkeys  were  not  successful. 

3.  The  monkey  may  pass  through  an  attack  of  typhus  so  mild 
that  it  cannot  be  recognized  clinically.     Vaccination  results. 

4.  The  immunity  test  is  a  reliable  proof  of  the  previous  occur- 
rence or  non-occurrence  of  typhus  at  least  within  a  period  of  one 
month. 

5.  Typhus  was  transmitted  to  the  monkey  by  the  bite  of  the 
louse  in  two  experiments,  the  lice  in  one  instance  deriving  their 
infection  from  man  and  in  another  from  the  monkey. 

6.  Another  monkey  was  infected  by  typhus  through  the  intro- 
duction of  the  feces  and  abdominal  contents  of  infected  lice  into 
small  incisions. 

Other  experiments,  in  which  the  immunity  tests  have  not  yet 
been  given,  corroborate  the  carrying  power  of  the  louse. 


472  Contributions  to  Medical  Science 

As  to  whether  these  experiments  on  transmission  are  to  be 
considered  merely  as  substantiating  that  of  Nicolle,  or  whether 
they  should  be  regarded  as  new  observations,  will  depend  on  the 
identity  or  non-identity  of  tabardillo  with  the  Old  World  typhus. 
If  Macacus  rhesus  is  susceptible  to  the  direct  injection  of  blood 
from  patients  suffering  from  tabardillo,  and  not  to  the  injection 
of  the  blood  of  the  Old  World  typhus,  the  two  diseases  in  our 
judgment  cannot  be  regarded  as  identical,  although  they  may  be 
closely  related.  As  stated  in  a  previous  paper,  Nicolle  was  unable 
to  infect  the  macacus  directly  with  human  blood,  but  succeeded 
first  in  infecting  the  chimpanzee  and  then  the  macacus  by  the 
injection  of  blood  from  the  chimpanzee.  Nicolle's  result,  if  it 
is  to  be  regarded  as  final,  when  compared  with  ours  and  with 
those  of  Anderson  and  Goldberger,  seems  to  indicate  non-identity 
of  the  disease  in  the  two  locaKties. 

It  would  seem  that  Nicolle's  work,  at  least  in  this  regard, 
demands  repetition.  Possibly,  also,  the  point  may  be  elucidated 
further  by  determining  whether  an  attack  of  Old  World  typhus 
confers  immunity  to  tabardillo. 

We  are  greatly  indebted  to  Director  Gavino  of  the  Bacteriologic  Institute,  to 
his  assistant,  Dr.  Girard,  to  their  superiors  of  the  Department  of  Public  Instruction, 
and  to  the  authorities  of  the  General  Hospital  of  the  City  of  Mexico,  in  particular  to 
Dr.  Escalona,  for  their  numerous  courtesies  and  co-operation. 


THE  ETIOLOGY  OF  THE  TYPHUS  FEVER  (TABARDILLO) 
OF  MEXICO  CITY. 

A  FURTHER  PRELIMINARY  REPORT.' 

H.    T.     RiCKETTS    AND    R  XJ  S  S  E  L  L     M.    WiLDER. 

{From  the   University  of  Chicago   [Department  of  Pathology]  and  the  Memorial  Institute  for  Infectious 

Diseases,  Chicago.) 

Some  months  ago^  we  reported  an  experiment  in  which  the  virus 
of  typhus,  as  it  exists  in  the  diluted  serum  of  the  patient,  failed 
to  pass  through  a  Berkefeld  filter.  The  monkey  which  received 
the  unfiltered  serum  exhibited  a  severe  course  of  fever  after  an 
incubation  period  of  five  days,  whereas  the  animal  which  received 
the  filtered  serum  developed  no  fever  and  remained  perfectly  well. 

The  immunity  test,  which  has  not  been  reported  heretofore, 
confirmed  the  conclusion  that  the  virus  did  not  pass  through  the 
filter.  This  test,  which  was  given  about  one  month  following  the 
"filtration  experiment,"  consisted  of  the  intraperitoneal  injection 
of  7  c.c.  of  diluted  defibrinated  blood  drawn  from  a  human  patient 
on  the  tenth  or  eleventh  day  of  his  fever.  No.  3,  which  had 
tolerated  the  filtered  serum  without  visible  disturbance,  showed 
a  course  of  high  fever  lasting  eleven  days,  and  preceded  by  an 
incubation  period  of  seven  days,  as  the  result  of  the  immunity 
test.  The  animal  which  had  received  the  unfiltered  serum  died 
as  a  consequence,  and  of  course,  no  immunity  test  could  be  given. 

This  experiment  has  been  repeated  with  a  similar  result.  No. 
19,  which  received  filtered  serum,  showed  no  signs  of  infection, 
whereas  No.  18,  which  was  given  unfiltered  serum,  passed  through 
the  customary  incubation  period  followed  by  a  severe  course  of 
fever. 

Anderson  and  Goldberger^   obtained  a  similar  result. 

It  seems,  therefore,  that  the  evidence  is  sufficiently  strong  that 
the  virus  of  tabardillo  does  not  pass  through  a  filter  of  the  type 
mentioned.    This  being  the  case,  the  result  may  be  taken  as  oflfer- 

'  From  Jour.  Am.  Med.  Assn.,  1910,  S4,  P-  1373- 

'  Ibid.,  p.  463;   p.  45 1  this  book. 

'Anderson  and  Goldberger,  Pub.  Health  Rep.,  February  18,  ipio. 

473 


474  Contributions  to  Medical  Science 

ing  a  suggestion  in  regard  to  the  probable  or  approximate  size 
of  the  virus.  It  seems  not  unhkely  in  view  of  its  non-filterability 
that  the  micro-organism  is  of  such  size  that  it  should  be  suscep- 
tible to  observation  microscopically,  provided  its  tinctorial  affinities 
and  density  are  favorable. 

Prompted  by  this  probability,  we  have  been  engaged  in  a  careful 
search  of  the  blood  of  patients  for  the  presence  of  micro-organisms, 
correlating  our  findings  with  the  microbic  content  of  the  organs 
of  the  body  louse,  which  has  the  power  of  carrying  the  disease. 
It  will  be  apparent  that  the  results  do  not  prove  that  the  organism 
to  be  described  is  the  cause  of  typhus,  but  to  our  minds  they  have 
a  suggestive  value  which  is  sufficient  to  render  their  preHminary 
presentation  justifiable.     They  may  be  stated  briefly  as  follows: 

1.  In  the  stained  (Giemsa)  preparation  of  the  blood  of  patients, 
taken  on  from  the  seventh  to  the  twelfth  day  of  the  disease,  we 
invariably  have  found  a  short  bacillus  which  has  roughly  the 
morphology  of  those  which  belong  to  the  "hemorrhagic  septicemia 
group."  Usually  it  appears  to  stain  solidly,  but  on  minute  exami- 
nation an  unstained  or  faintly  stained  bar  is  seen  to  extend  across 
the  middle.  Occasionally  two  organisms  are  seen  end  to  end. 
Exact  measurements  have  not  been  made,  but  when  compared 
with  the  size  of  the  erythrocyte,  their  length  is  estimated  at  hardly 
more  than  two  micromillimeters,  and  their  diameter  at  about 
one-third  this  figure.  Certain  other  bodies,  the  identity  of  which 
is  not  so  clear,  may  represent  degeneration  or  involution  forms 
of  the  above.  They  consist  of  two  stained  granules,  connected 
by  an  "intermediate  substance,"  which  is  stained  faintly  blue  or 
not  at  all.  Frequently  one  of  these  granules,  or  "poles,"  is  larger 
than  the  other  and  stained  a  deep  purple,  whereas  the  smaller 
takes  a  faint  blue  color. 

2 .  In  moist  preparations  of  the  blood  of  patients,  bacillary  bodies, 
with  a  structure  Hke  that  mentioned  above,  have  been  encountered 
in  all  cases.  The  differentiation  of  the  forms  into  two  halves, 
separated  by  a  line  or  narrow  zone  of  a  substance  of  different 
refractive  power,  may  be  observed.  They  possess  no  active 
motihty,  but  vibrate  more  or  less  rapidly. 

3.  The  dejecta  and  various  organs  of  a  large  series  of  lice  have 


Etiology  of  the  Typhus  Fever  of  Mexico  City     475 

been  stained  in  a  similar  way  and  examined  for  the  presence  of 
micro-organisms.  Certain  groups  had  been  deUberately  infected 
by  permitting  the  Hce  to  feed  on  patients,  while  others  were  sup- 
posedly normal,  having  been  collected  from  healthy  individuals. 
Streptococci,  staphylococci,  an  oval  bacillus  occurring  in  clusters, 
and  certain  solid  staining  bacilli  are  encountered  irregularly  and 
indifferently  in  the  feces  and  intestinal  contents  of  both  "normal" 
and  "infected"  lice.  Polar-staining  organisms  have  been  found 
occasionally  in  the  feces,  and  intestinal  contents  of  "normal" 
lice,  whereas  they  are  present  almost  constantly,  and  often  in 
large  numbers,  in  similar  material  from  "infected"  individuals. 

Protozoa  have  not  been  recognized.  Micro-organisms  are 
encountered  occasionally  in  preparations  of  the  salivary  glands, 
ovary,  eggs,  and  testes  of  the  louse.  Further  study  is  needed  to 
determine  whether  they  are  present  in  these  tissues  as  a  result  of 
faulty  technic,  or  not. 

TECHNIC. 

Probably  no  field  of  microscopic  research  is  more  exposed  to 
errors  and  misinterpretation  than  the  search  of  the  blood  for 
micro-organisms  of  an  unknown  character.  We  shall  not  at  this 
time  consider  the  many  sources  of  confusion  which  are  inherent 
in  the  blood  itself,  and  in  methods  of  its  preparation.  Naturally 
we  have  attempted  to  avoid  being  led  astray  by  these  conditions. 
Accidental  contaminations  from  outside  have  been  guarded  against 
carefully.  Slides  and  cover-glasses  were  cleaned  in  sulphuric  acid 
and  potassium  bichromate,  washed  in  freshly  distilled  water,  and 
preserved  in  absolute  alcohol.  The  latter  was  removed  by  freshly 
laundered  linen  just  prior  to  making  the  preparations.  Forceps 
previously  flamed  were  used  in  manipulating  the  glass.  The 
water  employed  in  diluting  the  Giemsa  stain  was  distilled,  then 
filtered,  autoclaved,  and  preserved  in  small  flasks,  which  had  been 
cleaned  and  rinsed  repeatedly  with  filtered  water.  Preparations 
of  the  blood  were  covered  at  once  to  avoid  contamination  from 
the  air.  The  blood  was  taken  from  the  ear  after  the  latter  had  been 
scrubbed  with  green  soap  and  absolute  alcohol  by  means  of  ster- 
ilized cotton. 


476  Contributions  to  Medical  Science 

frequency  of  the  bacilli  in  the  blood. 

The  organisms  described  have  been  found  to  be  more  numerous 
in  the  blood  within  three  or  four  days  prior  to  crisis  than  before 
this  period.  In  comparison  with  the  frequency  with  which  the  or- 
ganism of  malaria  and  trypanosomiasis  are  encountered  in  the  blood 
of  patients,  the  baciUi  would  be  considered  quite  rare.  It  is  rare 
to  recognize  a  bacillus  short  of  one  trip  across  a  three-fourths-inch 
cover-glass,  and  frequently  it  is  necessary  to  cover  this  distance 
two  or  three  times.  This  is  true  of  both  the  stained  and  the  fresh 
preparations.  A  rough,  and  naturally  inaccurate,  idea  of  their 
number  may  be  obtained  from  the  consideration  that  o.oi  c.c. 
of  blood  made  from  fourteen  to  twenty-four  smears  on  the  three- 
quarter-inch  cover-glass,  as  determined  in  two  observations.  With 
the  2-miUimeter  apochromatic  objective  of  Zeiss  and  with  the  No. 
6  compensating  ocular,  and  with  the  tube  drawn  out  to  i6  milli- 
meters it  is  required  to  traverse  the  preparation  from  80  to  90 
times  in  order  to  observe  the  entire  surface  of  one  cover-glass. 
Hence  one  might  expect,  roughly,  to  find  from  300  to  2,000  bacilH 
in  0.01  c.c.  of  blood. 

In  the  intestinal  contents  of  the  "infected"  louse,  it  is  occasion- 
ally necessary  to  search  for  three  or  four  minutes  before  bipolar 
organisms  are  found,  but  in  most  instances  organisms  of  this 
type  are  much  more  numerous  in  the  intestine  of  the  "infected" 
louse,  and  fifteen  or  twenty  may  be  found  in  a  single  field. 

It  has  occurred  to  us  that  the  segmented  bacilH  observed  in 
the  blood  may  be  identical  with  the  bipolar  forms,  which  are  found 
in  the  intestinal  contents  of  the  louse.  The  differences  are  not 
greater  than  those  which  are  encountered  in  the  known  organisms 
of  this  type,  such  as  the  plague  bacilli  and  that  found  in  association 
with  Rocky  Mountain  spotted  fever. 

THEORETICAL  CONSIDERATIONS. 

Considering  that  typhus  is  an  insect-borne  disease,  the  first 
thought  in  regard  to  its  microbic  etiology  would  naturally  involve 
an  organism  of  protozoan  character,  on  the  basis  of  analogies  which 
are  known  to  all.     Yet  our  knowledge  of  the  role  of  the  flea  in  the 


Etiology  of  the  Typhus  Fever  of  Mexico  City      477 

transmission  of  the  bacillus  of  plague,  of  the  tick  in  South  Africa 
in  carrying  the  spirillum  of  tick  fever,  and  of  the  same  insect  in 
carrying  the  bacilli  which  are  associated  with  Rocky  Mountain 
spotted  fever,  should  leave  us  without  prejudice  regarding  the 
etiology  of  some  other  insect-borne  disease.  It  seems,  therefore, 
that  our  minds  should  be  open  to  conviction  in  relation  to  a  bac- 
terial cause  for  typhus  as  well  as  protozoan. 

There  are,  in  addition,  at  least  two  features  of  typhus  which 
would  suggest  a  bacterial  rather  than  a  protozoan  cause. 

The  first  consists  of  the  fact  that  typhus  is  an  acute  self-limited 
disease,  in  so  far  as  clinical  evidence  can  show.  This  condition 
is  more  in  harmony  with  a  bacterial  rather  than  a  protozoan  cause, 
inasmuch  as  the  protozoan  diseases,  in  so  far  as  their  etiology  has 
been  determined,  are  chronic  in  character,  provided  the  animal 
survives  the  initial  acute  attack. 

A  second  condition  which  suggests  bacterial  etiology  consists 
of  the  fact  that  one  attack  of  typhus,  at  least  in  the  monkey, 
confers  immunity  to  further  inoculations.  It  is  true  that  not  all 
bacterial  diseases  confer  such  immunity,  but  it  is  equally  true  that 
immunity  is  not  recognized  as  a  distinctive  feature  of  protozoan 
infections. 

If  we  may  be  permitted  to  resort  to  another  analogy,  some 
ground  may  be  found  for  the  theory  that  typhus  is  caused  by  an 
organism  of  the  "hemorrhagic  septicemia  group"  of  bacteria. 
This  relates  to  the  fact  that  typhus  actually  is  a  hemorrhagic 
septicemia  from  the  clinical  and  experimental  standpoints,  and 
that  it  has  certain  points  which  are  distinctly  common  to  plague 
and  Rocky  Mountain  spotted  fever,  both  of  which  may  be  classed 
as  hemorrhagic  septicemias.  We  are  inclined  to  consider,  there- 
fore, that  the  three  diseases  mentioned  constitute  a  group  of  human 
"hemorrhagic  septicemias." 

We  are  aware  that  others  have  described  organisms  more  or 
less  similar  to  the  one  we  have  considered  in  relation  to  typhus, 
but  in  so  far  as  we  know  they  are  organisms  which  are  susceptible 
to  cultivation  by  ordinary  means.  Gavino  and  his  assistants  in 
Mexico  City  and  Anderson  and  Goldberger  have  failed  to  cultivate 
an  organism  of  any  type  from  typhus,  and  this  has  been  our  expe- 


478  Contributions  to  Medical  Science 

rience  also.  We  consequently  conclude  that  the  organism  we 
described  is  not  susceptible  to  cultivation  under  ordinary  condi- 
tions. This  is  corroborated  by  the  fact  that  two  experiments 
failed  to  yield  an  organism  of  this  type  from  the  intestinal  con- 
tents of  the  louse. 

It  is  the  purpose  of  this  paper  to  present  the  observations 
and  the  theoretical  considerations  which  have  been  mentioned 
solely  because  of  their  suggestive  value.  It  is  clear  in  our  minds 
that  the  grounds  are  not  sufficient  for  claiming  an  etiologic  role 
on  the  part  of  the  organism  described,  yet  the  conditions  under 
which  they  are  found,  together  with  the  theoretical  argument 
presented  above,  appear  to  demand  that  they  be  taken  somewhat 
seriously  and  subjected  to  further  study  in  their  relationship  to 
typhus. 

We  desire  to  express  our  obligations  to  the  Department  of  Public  Instruction  of 
Mexico,  to  Director  Gavino  of  the  Bacteriologic  Institute,  to  the  authorities  of  the 
General  Hospital,   and  particularly  to  Dr.  Escalona,  for  their  numerous  courtesies. 


THE  RELATION  OF  TYPHUS  FEVER  (TABARDILLO)  TO 
ROCKY  MOUNTAIN  SPOTTED  FEVER.^ 

H.     T.     RiCKETTS    AND    RuSSELL    M.     WILDER. 

(From  the  Department  of  Pathology,  University  of  Chicago,  and  the  Memorial  Institute  for  Infectious 
Diseases,  Chicago.) 

One  who  has  seen  Rocky  Mountain  spotted  fever  cannot  fail 
to  be  impressed  with  certain  points  of  similarity  which  the  disease 
shows  to  typhus  fever  (Typhus  exanthematicus) ,  basing  the  com- 
parison on  the  descriptions  of  typhus  which  are  given  in  standard 
treatises.  These  descriptions  refer  to  typhus  as  it  occurs  in  certain 
European  and  Asiatic  countries.  It  seemed  desirable,  therefore, 
to  study  their  relationship,  along  chnical,  anatomical,  and  immuno- 
logical Hues,  at  least  in  certain  essential  respects. 

Our  observations  concern  tabardillo,  the  typhus  fever  of  the 
great  Mexican  plateau,  which  differs  in  some  important  respects 
from  European  typhus,  according  to  the  opinion  of  those  who 
have  studied  the  disease  minutely  (e.g.,  Jose  Terres,  in  "Etiologia 
del  Tabardillo").  It  is  stated  that  the  typhus  of  Mexico  has  a 
more  gradual  onset  and  defervescence  than  that  of  the  Old  World. 
These  are  both  said  to  be  very  sudden  in  the  latter,  whereas  in 
the  former  the  fever  rises  gradually  for  three  or  four  days  during 
the  onset,  and  defervescence  occupies  a  similar  period.  The 
two  diseases  should  again  be  subjected  to  a  close  comparative 
study  in  these  respects. 

A  peculiar  topographic  distinction  exists  between  the  two,  in 
that  the  typhus  of  Mexico  is  Hmited  to  the  plateau  and  is  said  not 
to  occur  at  or  near  sea-level;  that  is  to  say,  it  does  not  occur  in 
the  so-called  "hot  country"  of  Mexico.  European  typhus,  on 
the  other  hand,  finds  its  home  to  a  large  extent  at  sea-level.  In 
view  of  the  fact,  however,  that  typhus,  the  world  over,  is  a  disease 
of  temperate  and  cool  climates,  the  discrepancy  mentioned  is 
only  an  apparent  one;  it  loses  significance  when  we  consider  that 
the  climate  of  the  great  Mexican  plateau  is  a  temperate  one,  while 

"  From  Arch.  Int.  Med.,  1910,  s,  p.  361. 

479 


480  Contributions  to  Medical  Science 

that  of  the  sea  coast  towns  is  warm  or  torrid,  in  which  general 
experience  indicates  that  typhus  is  not  able  to  prevail. 

comparison  of  spotted  fever  and  typhus  fever. 

Eruption. — In  both  spotted  fever  and  typhus  fever  a  macular 
or  slightly  elevated  roseolar  eruption  occurs,  which  commonly 
becomes  petechial,  and  which  appears  at  about  the  same  time 
in  both  diseases,  but  perhaps  a  little  earlier  in  spotted  fever.  In 
typhus  it  is  first  seen  at  about  five  days  after  the  beginning  of  the 
fever,  and  in  spotted  fever  on  from  the  second  to  the  fifth  day. 
In  both  diseases  small  hemorrhages  (petechiae)  may  occur  either 
in  the  pre-existing  rose-colored  spots  or  at  points  in  the  skin  which 
were  hitherto  uninvolved.  In  the  former  case  a  petechia  with  a 
congested  zone  is  formed,  and  in  the  latter  the  areola  is  absent. 
The  hemorrhages  appear  to  occur  earlier  and  with  greater  regu- 
larity in  tabardillo  than  in  spotted  fever,  although  this  phenome- 
non is  subject  to  great  variations  in  the  latter  disease.^  In 
some  instances  of  spotted  fever  sharply  marked  petechiae  do  not 
occur  at  all,  while  the  opposite  extreme  occasionally  is  encountered, 
the  petechiae  appearing  before  a  roseolar  eruption  is  definitely 
recognizable.  Although  petechiae  do  not  occur  invariably  in 
typhus,  they  would  seem  to  be  more  constant  than  in  spotted 
fever.  In  both  diseases  the  "spots"  show  a  certain  slight  degree 
of  induration;  this  is  quite  marked  in  some  cases  of  spotted  fever. 

There  seems  to  be  a  characteristic  difference  in  the  regions  of 
the  body  first  involved.  In  spotted  fever  the  spots  first  appear  on 
the  forearm  and  lower  leg  in  a  large  percentage  of  the  cases,  whereas 
in  typhus  they  are  first  seen  on  the  abdomen  and  sides  of  the  chest. 
There  are  variations,  however,  in  the  sequence  of  distribution  in 
spotted  fever,  so  that  it  is  doubtful  if  this  point  is  of  great  distinc- 
tive value. 

In  both  infections  the  distribution  in  the  end  is  a  very  general 
one,  including  the  face,  palms,  and  soles:  it  would  seem  that  the 
involvement  of  the  palms  and  soles  is  more  prominent  in  spotted 
fever  than  in  typhus.  As  regards  profuseness  there  is  no  essential 
difference. 

'  See  the  Fourth  Biennial  Report  of  the  Slate  Board  of  Health  of  Montana,  1907-8,  p.  137- 


Relation  of  Typhus  Fever  to  Spotted  Fever       481 

Gangrene. — In  the  spotted  fever  of  Idaho,  gangrene  of  the 
foreskin  and  scrotum,  the  tonsils  and  faucial  pillars  occurs  not 
infrequently;  this  is  not  seen  so  frequently  in  the  more  severe 
type  which  prevails  in  Montana.  In  typhus,  the  toes,  feet,  and 
lower  leg  occasionally  become  gangrenous,  and  extensive  bedsores 
are  rather  frequent. 

Changes  in  internal  organs. — In  spotted  fever  the  spleen  is 
habitually  enlarged.  This  can  be  determined  clinically  in  practi- 
cally all  cases;  and  at  autopsy  the  mass  of  the  spleen  is  sometimes 
three  or  four  times  that  of  the  normal  organ.  In  Mexican  typhus 
it  shows  Httle  enlargement  and  this  can  rarely  be  detected  cHnically. 
In  one  autopsy  it  was  of  normal  size;  in  another  it  was  slightly 
enlarged,  but  cirrhosis  of  the  liver  was  also  present.  In  both 
diseases  it  is  of  rather  firm  consistence,  in  no  way  resembling  the 
spleen  in  typhoid  fever. 

In  spotted  fever  the  lymph  glands  are  distinctly  but  moderately 
enlarged;  in  typhus  they  are  smaller,  but  probably  a  little  larger 
than  normal.     They  show  more  congestion  in  spotted  fever. 

In  typhus  the  meninges  almost  constantly  show  a  great  deal  of 
congestion  and  edema  at  autopsy,  so  that  it  has  sometimes  been 
spoken  of  as  meningeal  typhus.  This  condition  also  is  stated  as 
being  present  in  European  typhus,  but  perhaps  not  as  constantly 
as  in  that  of  Mexico.     It  is  a  minor  finding  in  spotted  fever. 

At  autopsy  the  right  heart  and  venous  system  show  more 
engorgement  in  spotted  fever  than  in  typhus,  and  this  corresponds 
also  with  the  clinical  appearances  of  the  two  diseases. 

Other  organs  appear  uninvolved  in  typhus  and  spotted  fever, 
except  for  the  presence  of  occasional  complicating  infections, 
particularly  pneumonia. 

There  appears  to  be  nothing  unique,  therefore,  as  to  anatomical 
changes  in  either  disease. 

Fe^er. — In  typhus  the  fever  begins  and  ends  with  a  good  deal  of 
abruptness.  Two  or  three  days  may  be  required  after  onset  before 
it  reaches  its  high  point,  and  an  equal  period  is  occupied  in  defer- 
vescence. On  the  other  hand,  the  temperature  in  spotted  fever 
may  not  reach  its  maximum  until  a  week  or  more  after  onset,  and 


482  Contributions  to  Medical  Science 

defervescence  may  occupy  a  week  or  ten  days.  This  is  one  of  the 
marked  differences  between  the  two  diseases. 

Pulse. — In  the  early  part  of  the  course,  and  in  mild  cases 
throughout,  the  pulse  in  spotted  fever  (90-110)  is  slower  than  in 
typhus  (i  10-120)  under  similar  conditions.  In  both  it  rises  to 
140  or  more  preceding  a  fatal  issue. 

Metital  effects. — Stupefaction,  or  a  low  nervous  delirium,  is 
common  to  both. 

Convalescence. — This  varies  with  the  severity  of  the  infection  in 
both  diseases,  but  on  the  whole  it  is  much  slower  in  spotted  fever 
than  in  typhus.  This  may  be  due  in  part  to  the  longer  duration 
of  spotted  fever. 

Duration. — The  "crisis"  in  typhus  usually  occurs  on  from  the 
tenth  to  the  fourteenth  day,  although  some  cases  may  cover  a 
period  of  three  w^eeks.  Patients  suffering  from  spotted  fever  are 
rarely  convalescent  until  the  end  of  the  third  week,  and  they 
commonly  remain  bedfast  for  from  four  to  six  weeks. 

Character  of  the  infections. — In  both,  the  condition  is  that  of 
a  systemic  blood  infection  (and  presumably  lymph  infection), 
without  the  critical  involvement  of  particular  organs.  These 
points  are  brought  out  by  the  findings  at  autopsy,  and  by  the 
result  of  inoculations  with  the  blood  of  patients.  Blood  from 
spotted  fever  patients  is  always  infective  for  the  guinea-pig,  mon- 
key, and  certain  other  animals.  In  a  number  of  instances  reported 
in  the  literature,  the  blood  of  tophus  patients  apparently  has 
produced  the  disease  when  injected  into  other  human  beings,  and 
NicoUe's  infection  of  the  chimpanzee  was  done  by  the  same  means ; 
this  concerns  European  typhus.  The  experiments  with  Mexican 
typhus,  reported  recently  by  Anderson  and  Goldberger  and  by 
ourselves,  also  show  that  the  latter  is  a  generalized  infection. 

Transmission. — Spotted  fever  is  not  contagious,  and  the  evi- 
dence indicates  that  the  same  is  true  of  typhus.  The  former  is 
transmitted  by  the  bites  of  certain  species  of  ticks,  while  presumably 
the  latter  is  carried  by  the  body  louse  (Pediculus  vestimenti). 
It  is  probable,  therefore,  that  they  have  the  feature  of  insect 
transmission  in  common,  but  two  altogether  different  types  of 
insects  are  concerned. 


Relation  of  Typhus  Fever  to  Spotted  Fever       483 

It  is  a  peculiar  fact  that  the  conception  of  contagiousness  has 
adhered  to  typhus  up  to,  and  including,  the  present  time.  Yet, 
in  view  of  the  facts  that  typhus,  when  endemic  in  a  city,  remains 
rather  strictly  segregated  in  the  poor  quarters,  and  that  more  or 
less  intimate  contact  is  required  for  transmission,  it  is  manifest  that 
contagiousness,  if  present  at  all,  must  be  of  a  peculiar  character 
and  of  a  low  grade.  Typhus  has  never  overwhelmed  a  whole  city 
as  smallpox  did  again  and  again  in  former  times.  In  recent  years, 
however,  behef  in  the  theory  of  insect  transmission  of  typhus  has 
extended  widely,  as  affording  a  better  explanation  of  the  epidemio- 
logic features  of  the  disease.  Thus  the  flea  and  bedbug  have 
repeatedly  been  mentioned  in  relation  to  European  typhus,  and 
Gavino  and  others  have  called  attention  to  the  possibihty  of 
insect  transmission  in  Mexico,  without  indicating  the  probable 
species,  however. 

The  recent  experiments  of  Nicolle  in  transmitting  European 
(rather  Asiatic)  typhus  from  monkey  to  monkey  by  means  of  the 
body  louse  affords  a  good  working  basis  for  clearing  up  the  natural 
means  of  transmission. 

Our  own  experiments  with  the  louse,  which  have  been  successful 
in  a  measure,  will  be  reported  at  a  future  date. 

Susceptibility  of  animals. — Aside  from  the  discrepancies  between 
spotted  fever  and  typhus  which  were  mentioned  above,  a  striking 
difference  is  found  in  the  susceptibility  of  animals  to  the  two 
diseases.  As  regards  European  typhus  the  literature  contains 
numerous  references  to  attempts  to  infect  the  guinea-pig  and  other 
ordinary  animals  of  experimentation  by  the  injection  of  blood 
from  patients,  the  results  being  uniformly  negative.  Likewise, 
in  extensive  experimentation  with  the  typhus  of  Mexico,  Director 
Gavino  of  the  Bacteriologic  Institute  of  Mexico  City,  and  his 
assistants,  failed  to  produce  any  evidence  of  infection  in  guinea- 
pigs,  rabbits,  white  rats,  and  mice  by  the  subcutaneous  and 
intravenous  injection  of  blood  from  human  patients,  the  blood 
being  taken  at  different  periods  of  the  disease.^  Similarly,  and 
in  substantiation  of  Dr.  Gavino's  results,  Anderson  and  Goldberger 

■  General  reference  to  these  experiments  is  made  in  an  article  by  Dr.  Gavino  in  Gaceta  Midica  de 
Mexico,  1906,  1,  p.  218.  They  are  also  cited  elsewhere  in  the  same  publication,  the  exact  references 
not  being  before  us  at  this  moment. 


484  Contributions  to  Medical  Science 

reported  their  failure  to  infect  the  guinea-pig  by  the  injection  of 
virulent  typhus  blood/ 

These  results  seem  so  conclusive  that  we  decided  not  to  repeat 
the  experiments. 

In  contrast  to  this  condition,  a  fairly  large  experience  has  shown 
that  spotted  fever  may  be  transmitted  to  the  guinea-pig  invariably 
by  the  subcutaneous  or  intraperitoneal  injection  of  virulent  blood, 
provided  no  serious  error  in  technic  has  been  made.^ 

The  difference  in  the  susceptibility  of  the  guinea-pig  to  the  two 
diseases  must  be  taken  as  showing  definitely  that  typhus  and 
spotted  fever  are  not  identical.  It  might  also  stand  as  sufficient 
reason  for  concluding  that  they  could  not  be  even  related  infec- 
tions, were  it  not  for  the  fact  that  there  are  two  types  of  spotted 
fever,  one  of  which  appears  to  be  less  virulent  for  the  guinea-pig 
than  the  other.  The  spotted  fever  of  western  Montana,  which 
represents  the  more  virulent  type,  can  be  maintained  indefinitely 
in  the  guinea-pig  by  passage  from  one  animal  to  the  other.  On 
the  other  hand,  it  has  been  impossible,  on  three  occasions,  by  the 
use  of  the  same  method,  to  keep  alive  in  the  guinea-pig  the  mild 
type  of  the  disease  which  prevails  in  southern  Idaho.  It  "died 
out"  after  from  two  to  ten  passages,  presumably  because  of  a  loss 
of  virulence  for  the  guinea-pig.  Yet,  other  experiments,  particu- 
larly that  of  agglutination,  as  performed  with  the  bacilH  found  in 
the  eggs  of  the  tick,  indicate  that  the  two  types  which  occur  in 
Montana  and  Idaho,  respectively,  are  identical  or  closely  related. 

As  bearing  on  typhus  fever,  this  condition  raises  the  question 
as  to  whether  there  may  be  a  third  type  of  infection  (typhus  fever) 
which  is  related  to  spotted  fever,  but  which  differs  from  it  not 
only  in  certain  important  clinical  respects,  but  also  in  possessing 
even  a  less  degree  of  virulence  for  the  guinea-pig  than  the  mild 
spotted  fever  referred  to  above.  In  other  words,  may  typhus 
fever  have  a  group  relationship  to  the  spotted  fever  of  the  Rocky 
Mountains  ? 

'Anderson  and  Goldberger,  "The  Relation  of  Rocky  Mountain  Spotted  Fever  to  the  Typhus  Fever 
of  Mexico — A  Preliminary  Note,"  Pub.  Health  Rep.,  1909,  24,  1861. 

'It  is  important  that  the  blood  injected  should  be  drawn  rather  early  in  the  disease,  and  that  the 
quantity  injected  should  not  be  too  large;  dilution  with  salt  solution  favors  infection.  This  has  been 
referred  to  in  previous  articles  by  one  of  us. 


Relation  of  Typhus  Fever  to  Spotted  Fever       485 

immunization  experiments. 

We  have  resorted  to  protective  and  agglutination  experiments 
in  order  to  obtain  more  conclusive  data  regarding  this  point.  The 
experiments  may  be  reported  briefly. 

One  attack  of  spotted  fever,  or  of  typhus,  renders  the  individual 
immime  to  further  attacks  of  the  same  disease.  This  acquired 
immunity  in  spotted  fever  is  characterized  by  the  formation  of 
protective  antibodies,  which  appear  in  the  blood,  and  which  can 
be  demonstrated  by  experiments  on  the  guinea-pig.  From  o.i 
to  o .  2  c.c.  of  serum  from  the  immune  guinea-pig  protects  against 
i.o  c.c.  of  virulent  blood,  representing  from  200  to  1,000  patho- 
genic doses.  From  0.3  to  0.5  c.c.  of  serum  from  the  convalescent 
human  patient  were  required  to  exert  the  same  protective  effect 
in  two  experiments.  If  typhus  were  identical  with  spotted  fever, 
the  serum  from  typhus  convalescents  should  exert  a  similar  pro- 
tective effect  against  spotted  fever;  or,  if  typhus  occupied  a 
"group  relationship"  to  spotted  fever,  this  might  be  manifested 
by  a  certain  (perhaps  low)  degree  of  protective  effect  against 
spotted  fever,  on  the  part  of  serum  from  typhus  convalescents. 

As  will  appear,  the  experiments  to  be  reported  do  not  disclose 
with  certainty  the  relationship  which  has  been  suggested,  and 
possibly  they  are  of  such  value  that  a  close  relationship  is  actually 
disproved. 

The  immune  typhus  serums  were  all  taken  from  patients 
whose  course  we  had  observed  in  the  General  Hospital  (Mexico 
City),  and  in  whom  the  diagnosis  of  typhus  seemed  to  be  with- 
out doubt.  Their  histories  will  not  be  recited.  The  blood  was 
drawn  on  from  the  seventh  to  the  tenth  day  after  the  sub- 
sidence of  fever,  after  the  patients  had  left  their  beds,  and  the 
serum  obtained  by  defibrination  and  centrifugation  or  by  spon- 
taneous clotting. 

Three  sets  of  controls  were  utilized:  first,  the  protective  power 
of  normal  human  serum  as  compared  with  that  from  the  typhus 
convalescents;  second,  the  tests  of  the  toxicity  of  human  serum 
alone  for  the  guinea-pig;  third,  inoculations  to  determine  approxi- 
mately the  strength  (quantity  or  virulence)  of  the  spotted  fever 
virus  used  in  each  experiment.    The  virus  was  that  represented  in 


486 


Contributions  to  Medical  Science 


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Relation  of  Typhus  Fever  to  Spotted  Fever       487 

the  defibrinated  blood  of  the  infected  guinea-pig  on  the  third 
day  of  its  fever. 

The  virus  and  serums  were  mixed,  and  injected  intraperitoneally 
as  soon  as  possible  thereafter;  the  syringe  was  washed  with  salt 
solution  and  the  washings  injected,  in  order  to  render  the  experi- 
ments as  nearly  quantitative  as  possible. 

It  will  be  noted  that,  as  the  experiments  progressed,  the  pro- 
portion of  convalescent  typhus  serum  to  spotted  fever  virus  in- 
creased from  ^  to  I  (experiment  i),  to  200  to  i  (experiment  4). 

One  would  expect  to  find  evidence  of  a  protective  power  in 
absolute  prevention  of  infection,  or,  if  present  to  a  low  degree, 
in  a  prolongation  of  the  incubation  period  above  that  of  the  con- 
trols, in  a  shorter  course  of  fever,  or  in  recovery  as  compared  with 
the  death  of  the  controls.  Three  animals  in  the  series,  9a,  iia, 
and  12a,  suffered  such  light  attacks  that  they  could  not  be  recog- 
nized positively  by  the  temperatures  exhibited,  the  result  being 
determined  only  by  immunity  tests  which  were  administered 
later.  The  accompanying  table  gives  the  outlines  of  the  experi- 
ments. 

Analysis  of  the  table  shows  with  reasonable  clearness  that  the 
typhus  serum  exerts  no  more  protective  effect  than  does  normal 
serum.  Interest  centers  chiefly  in  experiment  4,  in  which  the 
dose  of  virus  was  approximately  twice  the  minimum  infective 
quantity.  The  immune  typhus  serum  showed  a  certain  degree  of 
protection  in  doses  of  i.o  and  2.0  c.c,  but  on  the  other  hand, 
i.o  c.c.  of  normal  serum  showed  the  same  degree  of  protective 
power.  We  may  consider  that  the  dose  of  virus  used  was  so  low 
that  very  slight  influences  were  able  to  determine  the  occurrence 
or  non-occurrence  of  infection. 

Another  experiment  also  indicates  that  an  attack  of  typhus 
fever  in  the  monkey  does  not  protect  the  animal  against  a  subse- 
quent infection  with  spotted  fever  virus.  Monkey  1 1  was  inoculated 
with  typhus  by  means  of  blood  drawn  from  Monkey  7  on  the 
eighth  day  of  the  latter's  fever.  After  an  incubation  period  of 
nine  or  ten  days  the  temperature  of  No.  11  rose,  and  the  animal 
passed  through  a  course  of  fever  similar  to  that  which  has  appeared 
in  other  animals  inoculated  with  virulent  typhus  blood.     Twenty- 


488 


Contributions  to  Medical  Science 


seven  days  after  the  inoculation  and  after  recovery  was  complete 
a  second  injection  of  typhus  virus,  consisting  of  5  c.c.  of  blood 
from  a  human  patient,  was  given  to  No.  11,  in  order  to  determine 
immunity  or  non-immunity  to  the  disease.  No  fever  resulted 
over  a  period  of  eighteen  days.  A  control  monkey  (No.  21)  which 
received  a  similar  injection  passed  through  a  course  of  fever  resem- 
bling that  of  typhus.  We  may,  therefore,  consider  that  No.  11  had 
been  infected  by  its  first  injection  of  typhus  virus,  and  that  this 
resulted  in  immunity  to  the  disease. 

In  order  to  test  the  animal's  immunity  to  spotted  fever,  after 
the  lapse  of  some  days  it  was  injected  intraperitoneally  with  3 
c.c.  of  defibrinated  blood  from  the  infected  guinea-pig,  drawn  on 
the  third  day  of  the  latter's  fever.  The  blood  was  diluted  to  6  c.c. 
by  means  of  salt  solution  before  being  injected.  The  following 
course  of  fever  developed: 


A.M. 

P.M. 

A.M. 

P.M. 

March  13 

March  14 

March  15 

March  16 

March  17 

March  18 

102 

102,6 

102 

103 

104.7 

103. 1 

102.4 
103.3 
102.4 
104.6 
105.6 
106.4 

March  19 

March  20 

March  21 

!  March  22 

j  March  23 

March  24 

104.6 

104 

105.8 

105 

IOI.4 

99 

106.7 
105.6 
106.8 
106.6 
104.1 
I03 

On  March  23  and  24  an  extensive  hemorrhagic  eruption  devel- 
oped on  the  extremities,  tail  and  back,  such  as  has  occurred  pre- 
viously in  spotted  fever  in  the  monkey. 

As  a  control  a  guinea-pig  inoculated  with  i  c.c.  of  the  same 
virus  developed  a  typical  course  of  spotted  fever,  as  did  a  second 
guinea-pig  inoculated  with  the  blood  of  No.  11. 

This  experiment  indicates,  therefore,  that  an  attack  of  typhus 
in  the  monkey  does  not  render  him  immune  to  spotted  fever, 
although  it  does  protect  him  against  infection  from  a  second 
injection  of  virulent  typhus  virus. 

agglutination  experiments. 

As  reported  previously  by  one  of  us,  immune  spotted  fever 
serum  agglutinates  to  a  marked  degree  a  bacillus  which  occurs 
in  the  eggs  of  ticks  which  act  as  carriers  of  the  disease.  The 
agglutination  is  specific,  in  that  normal  serums  do  not  have  this 


Relation  of  Typhus  Fever  to  Spotted  Fever        489 

agglutinating  effect,  or  have  it  to  a  very  low  degree;  and  this, 
among  other  reasons,  is  taken  to  indicate  that  the  organism  bears 
a  causal  relation  to  spotted  fever. 

This  reaction  was  utilized  as  a  means  of  determining  a  possible 
relation  between  spotted  and  typhus  fevers.  As  in  previous 
experiments,  an  emulsion  of  the  bacilli  was  obtained  by  crushing 
a  sufficient  number  of  eggs  in  a  small  quantity  of  salt  solution, 
and  with  this  emulsion  the  microscopic  agglutination  test  was 
performed  with  various  dilutions  of  serum  from  convalescent 
typhus  patients.  Duphcate  preparations  with  normal  human 
serum  were  made,  and,  in  addition,  with  immune  spotted  fever 
serum  from  the  guinea-pig  in  order  to  make  certain  of  the  char- 
acter of  the  bacilli. 

In  the  case  of  the  immune  spotted  fever  serum  the  agglutination 
was  marked  or  complete  in  three  experiments  up  to  a  dilution 
of  I  in  500.  Numerous  experiments  have  shown  that  serum  from 
normal  guinea-pigs  agglutinates  only  in  dilutions  of  i  in  10  to 
I  in  20.  Normal  human  serum  caused  slight  agglutination  at  low 
dilutions  (i  in  10  and  i  in  20).  The  serum  of  typhus  convalescents, 
drawn  within  a  week  to  ten  days  after  the  subsidence  of  fever,  had 
approximately  the  same  agglutinating  power  as  the  normal  serum 
in  two  experiments,  while  in  a  third  it  reached  a  dilution  of  i  in 
40.  Even  in  this  case,  however,  the  agglutination  was  not  com- 
plete at  this  dilution. 

SUMMARY  AND   CONCLUSIONS. 

The  serum  of  typhus  convalescents,  drawn  within  a  week  to 
ten  days  after  the  subsidence  of  fever,  exerts  no  more  protective 
effect  against  spotted  fever  than  normal  serum  does. 

Also,  such  serums  show  little  or  no  more  agglutinating  effect 
for  the  bacilli  which  appear  to  be  associated  with  spotted  fever 
than  do  normal  serums. 

A  monkey  which  had  been  rendered  immune  to  typhus  was  not 
immune  to  spotted  fever. 

These  experiments  go  to  substantiate  the  clinical  evidence  and 
that  obtained  by  animal  inoculations,  that  spotted  fever  and 
typhus  fever  are  not  identical,  and  they  seem  also  to  indicate  that 


490  Contributions  to  Medical  Science 

the  organisms  of  the  two  diseases  are  not  closely  related  biologically, 
whatever  the  morphologic  conditions  may  be. 

The  fact  also  that  the  serum  of  a  disease  (typhus)  which  is 
roughly  similar  to  spotted  fever  has  no  unusual  agglutinating 
power  for  the  bacilli  mentioned,  as  compared  with  the  high  agglu- 
tinating power  of  immune  spotted  fever  serum,  supports  the  con- 
tention that  this  bacillus  bears  a  causal  relation  to  spotted  fever. 

We  are  indebted  to  Director  Gavino  of  the  Bacteriologic  Institute  and  to  his 
assistant,  Dr.  Girard,  for  the  use  of  their  laboratory  and  for  numerous  courtesies, 
to  Dr.  Escalona  of  the  General  Hospital  for  his  co-operation,  and  to  Mr.  J.  J.  Moore 
of  the  University  of  Chicago  for  assistance  in  the  experiments. 


FURTHER    INVESTIGATIONS    REGARDING    THE 
ETIOLOGY  OF  TABARDILLO,  MEXICAN 
TYPHUS  FEVER/ 

H.  T.  RiCKETTS  AND  RuSSELL  M.  WILDER. 

{Prom  the  Department  of  Pathology  of  the  University  of  Chicago  and  the  Memorial  Institute  for  Infectious 

Diseases,  Chicago.) 

In  a  previous  article^  we  have  reported  certain  experiments 
which  indicated  the  role  of  the  body  louse  (Pediculus  vestimenti) 
in  the  transmission  of  Mexican  typhus  fever.  Since  that  date  we 
have  been  able  to  confirm  the  results  of  our  first  investigations, 
and  also  to  obtain  certain  other  experimental  data  concerning  the 
etiology  of  this  disease.  The  experiments  which  we  wish  to  report 
at  present  relate  to  the  hereditary  transmission  of  the  infectivity 
of  the  louse  and  the  possible  role  of  the  bedbug  and  the  flea  in  the 
transmission  of  typhus. 

HEREDITARY    TRANSMISSION    OF    THE    INFECTIVITY    OF    THE    LOUSE. 

The  following  experiment  was  undertaken  with  a  view  to  deter- 
mining whether  the  young  of  infected  lice  were  themselves  infected. 
The  adult  louse  contains  in  its  ovaries  many  mature  eggs.  These 
eggs  are  covered  with  a  compact  shell  which  we  thought  might 
prove  impermeable  to  micro-organisms.  Hence  it  was  decided 
to  rear  young  lice  to  maturity  on  the  bodies  of  typhus  patients,  so 
that  if  the  eggs  were  susceptible  to  infection  at  any  stage  of  their 
development,  they  would  have  every  opportunity  of  being  infected 
within  the  ovary. 

On  March  29,  140  adult  lice  of  group  17  (Pediculus  vestimenti), 
70  males  and  70  females,  were  placed  in  a  stocking  on  the  leg  of  a 
typhus  patient  (No.  41).  The  stocking  was  sealed  above  with 
adhesive  tape  to  prevent  the  escape  of  any  of  the  insects.  Two 
days  later  i  ,000  eggs  were  found  adhering  to  the  fibers  of  the  stock- 
ing. The  lice  were  removed  and  replaced  on  the  patient  in  a  fresh 
stocking,  while  the  stocking  containing  the  eggs  was  put  on  the 


■  From  Jour.  Am.  Med.  Assn.,  igio,  55,  p.  309. 
'Ibid.,  54,  p.  1304.    See  p.  463  of  this  book. 


491 


492  Contributions  to  Medical  Science 

patient's  other  leg.  Approximately  800  more  eggs  were  subse- 
quently laid  by  this  generation  of  lice. 

By  April  6  many  of  the  eggs  began  to  hatch  and  by  April  15 
about  500  young  lice  had  been  collected.  These  were  placed  in  a 
fresh  stocking  which  was  kept  constantly  on  the  leg  of  a  patient 
in  an  early  stage  of  fever.  For  this  purpose  patients  47,  49,  50, 
52,  and  53  were  used.  Many  of  the  young  lice  died,  but  approxi- 
mately 250  of  them  reached  maturity  and  in  turn  laid  eggs. 

When  a  sufficiently  large  number  of  these  presumably  infected 
eggs  of  the  second  generation  had  been  obtained,  all  of  the  adult 
lice  were  removed  and  placed  in  a  new  stocking  on  the  same  patient. 
The  stocking  containing  the  eggs  was  then  sealed  and  incubated 
between  the  sheet  and  the  mattress  of  a  patient  in  an  early  stage 
of  convalescence. 

Monkey  42,  of  the  species  Macacus  rhesus,  who  served  for  this 
experiment,  had  been  recently  imported  into  Mexico  from  a  dis- 
trict free  from  typhus.  As  the  eggs  hatched,  the  young  lice  were 
collected  and  placed  on  this  normal  monkey  in  the  following 
manner.  The  animal's  skin  was  shaved  over  the  entire  abdomen 
and  a  piece  of  finely  woven  linen,  two  inches  by  three,  was  tightly 
secured  to  the  skin  by  means  of  a  border  of  two-inch  adhesive  tape. 
One  edge  of  the  cloth  was  left  unattached  and  the  open  end  of  a 
tube  containing  the  young  Hce  inserted  beneath  this  edge,  the  lice 
being  dumped  into  the  pocket  formed  between  the  cloth  and  the 
skin.  The  mouth  of  the  pocket  was  then  sealed  with  tape  and  the 
animal  clothed  in  a  heav>'  canvas  jacket  in  order  to  prevent  inter- 
ference with  the  lice.  Thus  on  April  28,  fifty  young  lice  were  placed 
on  the  monkey.  On  April  30,  thirty  more  were  added.  At  this  time 
it  was  noted  that  the  Hce  of  April  28  had  fed,  their  bodies  being 
gorged  with  blood.  On  May  2,  twenty-five  additional  lice  were 
collected  from  the  stocking  and  placed  on  the  monkey.  Thus  in 
all  105  lice,  the  offspring  of  infected  lice,  but  themselves  never  di- 
rectly infected,  were  given  an  opportunity  of  feeding  on  monkey  42. 

Unfortunately  the  temperature  of  this  animal  could  not  be 
taken  regularly  during  the  following  three  or  four  weeks,  nor  was 
he  under  very  careful  observation  during  this  period.  On  May 
26,  however,  he  appeared  to  be  in  good  health.     But  more  impor- 


Investigations  of  Mexican  Typhus  Fever 


493 


tant  is  the  fact  that  he  proved  resistant  to  a  subsequent  immunity 
test  described  below. 

In  our  work  on  transmission  with  Hce  we  have,  with  one  excep- 
tion, never  succeeded  in  provoking  a  very  characteristic  febrile 
reaction  in  the  macacus  monkey  by  means  of  the  bite  of  Hce,  the 
relative  insusceptibility  of  the  monkey  to  typhus  probably  account- 
ing for  his  resistance  to  infection  by  lice.  It  has  been  found, 
however,  that  monkeys  who  have  been  subjected  to  the  bites  of 
infected  lice  are  thereby  rendered  immune  to  subsequent  inocula- 
tions of  typhus  blood  which  constantly  provokes  in  normal  monkeys 
a  high  febrile  reaction.  This  immunity  has  been  interpreted  as 
indicating  that  the  animal  has  been  infected  by  the  Hce  and  has 
suffered  a  mild  attack  of  typhus,  and  the  immunity  test  thus  con- 
stitutes the  chief  criterion  for  determining  whether  or  not  an  animal 
has  been  infected  by  insects. 

Such  an  immunity  test,  consisting  of  the  intraperitoneal  inocu- 
lation of  3 . 5  c.c.  of  virulent  typhus  blood  from  a  typhus  patient 
(No.  58),  was  given  to  monkey  42  on  May  27.  For  the  following 
three  weeks  the  animal  remained  in  perfect  health,  although  con- 
trols inoculated  with  the  same  quantity  of  the  same  material  all 
contracted  a  typhus  of  moderate  severity,  their  temperatures 
maintaining  an  elevation  of  104  to  105 . 7  F.  for  a  period  of  ten  or 
eleven  days. 

TABLE  I. 
Immunity  Test  of  Monkey  42. 


May  28 
May  29 
May  30 
May  31 
June  I 
June  2 
June  3 
June  4 
June  5 
June  6 
June  7 
June  8 
June    9 


A.M. 


102.6 
101.6 
ICI.2 
101.6 
101.4 
101.6 
102.0 
101.6 
104. 1 
103.2 
103.0 
I01.6 
100.4 


P.M. 


102.2 
101.9 
101.9 
102.0 

101.8 
102.2 
102.4 
102.4 

103.7 
103.0 
102.8 
103.0 

102.2 


June  10 
June  II 
June  12 
June  13 
June  14 
June  IS 
June  16 
June  17 
June  18 
June  19 
June  20 
June  21 
June  22 


A.M. 


102.3 
102.0 
102.0 
100.8 
99.6 
100.3 
101.2 
100.9 
100.4 
101 .0 
100.8 
100.2 
101.3 


P.M. 


102.1 
102.  S 
102.8 
102.0 
100.6 

101. 5 
101.4 
101.9 
102.0 
102.2 
101 .7 
102.4 
101.8 


Table  i  shows  the  temperatures  of  monkey  42  on  the  successive 
days  foHowing  the  inoculation.  Table  2  those  of  the  control  monkey 
(No.  44)- 


494 


Contributions  to  Medical  Science 


TABLE  2. 
Temperature  of  Monkey  44  Following  Inoculation  with  Typhus  Blood. 

A.M. 

P.M. 

A.M. 

P.M. 

May  a8 

102.3 
102.0 
loi  .9 
loi.s 
100.6 
101.6 
102.6 
105.6 
104.0 
103 -3 
103.5 

102.5 

102.  s 
102.0 

102.3 

loi  .6 
103.7 
103-3 
105.7 
1 04 -3 
103.3 
104.9 

June    8 

June    9 

June  10 

June  II 

June  12 

June  13 

June  14 

June  IS 

June  16 

June  17 

104.0 
104.1 
105.6 
104.6 
103.2 
loi  .6 
loi  .0 

lOI.O 

100.6 
100.3 

105.5 

104.7 

103.9 

105.2 

103.1 

102. 1 

102.2 

101.6 

101.6 

lOI.O 

It  has  been  our  experience  to  find  a  great  variation  in  the  daily- 
temperatures  of  normal  macacus  monkeys.  These  animals  are 
prone  to  slight  intestinal  disorders  and  a  sporadic  elevation  of  one, 
or  even  two  degrees,  has  been  frequently  noticed.  The  normal 
afternoon  temperatures  of  many  of  our  monkeys  lay  constantly 
between  103°  and  104°  F.  Hence  there  arises  a  certain  difficulty 
in  interpreting  the  sHght  elevation  of  temperature  shown  by 
monkey  42  on  June  5  and  June  6.  This  may  or  may  not  be  a  mild 
efifect  of  the  virus,  but  in  any  case  the  effect  was  far  less  than  that 
obtained  before  in  all  normal  animals,  including  the  control  monkey 
(No.  44)  of  this  particular  experiment,  and  it  is  quite  probable  that 
the  elevation  observed  was  purely  accidental. 

We  appreciate  that  the  result  of  one  experiment  does  not  con- 
stitute decisive  proof,  but  the  definiteness  of  the  result  justifies 
us,  in  our  opinion,  in  concluding  that  monkey  42  owed  his  immunity 
to  his  previous  infection  by  the  yoimg  lice  of  group  17  and  that 
hereditary  transmission  of  the  infectivity  of  the  louse  is  established 
to  the  extent  of  reasonable  probability. 

infectiousness  of  the  flea  and  of  the  bedbug. 
Theoretical  considerations  make  it  seem  extremely  probable 
that  neither  the  bedbug  nor  the  flea  plays  any  role  in  the  trans- 
mission of  typhus.  We  have  called  attention  to  this  in  a  previous 
article,'  and  Anderson  and  Goldberger'  have  expressed  the  same 
opinion.  The  bedbug  is  only  rarely  carried  about  in  the  clothing 
and  yet  it  is  well  known  that  people  who  handle  the  clothing 

■  Jour.  Am.  Med.  Assn.,  February  s,  1910,  34,  p.  463.     See  p.  451  of  this  book. 
"  Pub.  Health  Rep.,  February  18,  1910,  No.  7. 


Investigations  of  Mexican  Typhus  Fever  495 

of  typhus  patients,  such  as  laundresses  and  servants,  are  fre- 
quently stricken  with  typhus.  A  number  of  cases  of  typhus  have 
occurred  during  the  last  year  among  the  nurses  and  servants  of 
the  typhus  pavilion  of  the  General  Hospital  of  Mexico  City.  A 
careful  search  failed  to  reveal  any  bedbugs  in  this  building. 
Furthermore,  if  the  bedbug  transmitted  typhus  we  should  expect 
the  disease  to  be  a  "house  disease";  but  cases  are  constantly 
occurring  which  it  is  impossible  to  trace  to  any  "typhus  house." 

The  flea  also  seems  innocent.  Did  it  play  any  role,  the  dis- 
tribution of  typhus  in  Mexico  City  would  be  much  more  general 
than  is  the  case,  for  the  flea  is  widely  distributed.  Typhus,  how- 
ever, is  confined  almost  exclusively  to  those  living  under  less  satis- 
factory hygienic  conditions.  Finally,  as  previously  mentioned,  the 
season  of  typhus  is  the  winter  and  early  spring,  whereas  the  period 
of  greatest  prevalence  of  the  flea  is  the  summer. 

In  order  to  throw  further  light  on  this  question  the  following 
experiments  were  undertaken. 

EXPERIMENTS   ON  THE   INFECTIOUSNESS   OF  THE   BEDBUG. 

A  group  of  about  fifty  bedbugs  was  fed  on  three  successive 
days  on  patients  at  the  General  Hospital.  The  bugs  were  confined 
beneath  a  small  wide-mouthed  glass  bottle  inverted  over  the  skin 
of  the  patient.  In  this  way  ten  or  twelve  could  be  fed  at  one  time. 
All  were  given  the  opportunity  of  gorging  themselves  with  blood. 

On  April  2,  two  days  after  their  last  feeding  on  a  patient,  they 
were  placed  on  the  shaved  abdomen  of  a  monkey  (No.  35)  and 
allowed  to  feed.  On  April  3  they  were  again  placed  on  the  monkey, 
only  eight  feeding.  On  April  4  they  were  not  fed.  On  April  5, 
twenty-eight  bugs  fed  well  on  the  animal;  April  6,  eighteen  bugs 
fed  Hghtly;  April  7,  seventeen  bugs  fed  lightly;  April  12,  29  bugs 
fed  well,  gorging  themselves  with  blood. 

The  animal  showed  absolutely  no  rise  in  temperature,  and 
continued  in  excellent  health  during  the  following  thirty-two  days. 
Unfortunately  he  died  from  an  accident  before  an  immunity  test 
could  be  given  him,  and  hence  the  experiment  cannot  be  taken  as 
proof  of  the  non-infectiousness  of  the  bedbug.  It  does  seem  to 
eliminate  the  possibiUty  that  the  bedbug  can  transmit  tophus  more 


496  Contributions  to  Medical  Science 

readily  than  can  the  louse,  in  so  much  as  both  the  period  of  feedings 
on  the  infected  host  and  the  period  of  feeding  on  the  monkey  were 
considerably  in  excess  of  the  feedings  of  the  louse  in  certain  of  our 
experiments. 

EXPERIMENTS   ON   THE   INFECTIVITY   OF   THE   FLEA. 

Human  fleas,  of  flea  group  2,  were  infected  by  repeated  feedings 
on  typhus  patients  at  the  General  Hospital  in  the  following  manner : 
One  or  two  fleas  were  confined  in  each  of  several  long  and  narrow 
tubes.  These  tubes  were  made  of  4  mm.  glass  tubing,  were  sealed 
at  one  end  and  cut  sufl&ciently  long  (20  cm.)  to  prevent  the  escape 
of  the  flea  by  jumping.  For  this  purpose  also  they  were  slightly 
bent  in  the  middle.  The  fleas  were  fed  by  inverting  this  tube  on 
the  patient's  skin. 

Subsequent  to  their  last  feeding  the  fleas  were  allowed  to  rest 
for  about  sixty  hours.  The  entire  bodies  of  ten  of  the  group  were 
then  emulsified  in  physiologic  salt  solution  and  rubbed  into  scari- 
fications of  the  abdominal  skin  of  a  normal  monkey  (No.  41),  the 
technic  employed  in  this  procedure  being  the  same  as  that  used 
in  the  scarification  experiment  performed  with  the  intestinal  con- 
tents of  lice  and  previously  reported.  The  wounds  healed  with 
but  little  suppuration.  The  animal's  temperature  was  taken  twice 
daily  for  the  following  thirty-four  days  and  during  this  time  he 
remained  in  perfect  health. 

On  May  27  this  monkey  was  given  an  immunity  test,  receiving 
an  inoculation  of  3 . 5  c.c.  of  typhus  blood  from  patient  58.  After 
an  incubation  period  of  seven  days  he  began  to  run  a  fever  which 
lasted  for  twelve  days,  his  temperature  being  recorded  in  table  3. 

On  June  4  a  leukocyte  count  was  made^  which  showed  30,350. 
The  animal  was  very  irritable  and  his  coat  dry  and  ruffled.  On 
the  5  th  he  had  diarrhea  and  until  the  14th  of  the  month  seemed  very 
sick.    His  subsequent  recovery  was  rapid  and  complete. 

In  brief.  No.  41  had  not  been  infected  by  the  fleas,  as  is  shown 

» Leukocytosis  is  observed  in  nearly  all  cases  of  typhus  in  man.  In  conjunction  with  Dr.  Francis 
E.  Prestley  of  Mexico  City,  we  have  made  a  series  of  leukocyte  counts  of  most  of  the  monkeys  in  our 
possession.  It  was  found  that  monkeys  sick  with  typhus  fever  usually  showed  a  marked  increase  in  the 
number  of  white  blood  corpuscles  over  the  normal  which  approximates  12,000.  Differential  counts  based 
on  the  same  material  will  be  reported  in  a  later  communication. 


Investigations  of  Mexican  Typhus  Fever 


497 


by  the  fact  that  he  was  not  immune  to  a  subsequent  inoculation  of 
typhus  blood. 

TABLE  3. 
Temperature  of  Monkey  41  after  Receiving  an  Immunity  Test. 


May  28 
May  29 
May  30 
May  31 
June  I 
June  2 
June  3 
June  4 
June  s 
June  6 
June  7 
June  8 
June    9 


A.M. 


102.6 
101.8 
102.3 
loi  .6 
loi  .9 
101.9 
101.4 
103. 1 
104.5 
103. 1 
105.0 
103.2 
roi.4 


P.M. 


102.6 
102.0 

102.8 

102.6 
IOI.9 
102.7 
102.3 
104. 1 
104-5 
104. 1 
105.3 
103.6 
103. 1 


June  10 
June  II 
June  12 
June  13 
June  14 
June  IS 
June  16 
June  17 
June  18 
June  19 
June  20 
June  21 
June  22 


A.M. 


104.4 
104.4 
103.6 
104.6 
103.5 
103.9 
102.9 
102.  s 
102.5 
loi.s 
101 .6 
loi.s 
loi  .9 


P.M. 


105.0 
105.6 
105.5 
104.8 
104.8 
103.5 
102.8 
102.8 
102.0 
101.8 
102.  s 
102.6 


These  results  seem  to  strengthen  our  previous  position  as  to  the 
unimportance  of  the  flea  and  the  bedbug  in  the  transmission  of 
Mexican  typhus  fever. 

We  take  pleasure  in  expressing  our  obligations  to  Dr.  Liceaga  and  his  assistants 
of  the  Superior  Board  of  Health  of  Mexico,  to  the  authorities  of  the  General  Hospital, 
and  to  Dr.  Gavino  of  the  Bacteriologic  Institute  for  numerous  courtesies.  In  partic- 
ular we  wish  to  acknowledge  our  indebtedness  to  Dr.  Genaro  Escalona,  Dr.  Francis 
E.  Prestley,  and  Sr.  D.  Perez  Garga  of  Mexico  City,  for  co-operation  and  assistance. 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 

Los  Angeles 
This  book  is  DUE  on  the  last  date  stamped  below. 


BiowiDpfS  1  4  REC'D 


c^.RtCC 


tSL 


NOV  ?l^^ 


2  WK  from  Receipt 

JUL  4    1974 

JI1.>SI»MLS 

JUM^2"Cr^Er&- 
MAR  2  4  REC'[^ 

BioMEo  MAR  2076 


DEC    ^®f 

Form  L9-100m-9,'52(A3105)444 


R4:i^C 


3   1158  OCpS  462P 


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